Chemical and Clinical Applications of Tempol: A Marvelous Molecule 9781032730028

Table of contents :
Cover
Half Title
Chemical and Clinical Applications of Tempol: A Marvelous Molecule
Copyright
Contents
Preface
Author biographies
Acknowledgments
1. Introduction to Tempol
Introduction
Mechanisms of nitroxide reactions with biologically relevant small radicals
Applications of tempol
Conclusion
References
2. Synthesis and Chemical Reactions of Tempol
3. Tempol in the Synthesis of Terpenoids
4. Name Reactions Involved in TEMPOL
Introduction
Machetti–De Sarlo reaction
Mannich reaction
TEMPOL Oxidation
TEMPOL Oxidation of benzylic alcohols
Sheldon synthesis of TEMPOL
Tempo-Mediated oxidation
Conclusion
References
5. Industrial Applications of TEMPOL
6. The Role of Tempol in NRTI-Induced Mitochondrial Toxicity
7. The Significance of Tempol in Diabetic Nephropathy
8. Mechanistic Insights into the Reaction Between a Nitroxide Radical (Tempol) and a Phenolic Antioxidant
Introduction
Structure of nitroxides
Reactions involving nitroxide radicals and antioxidants
Conversion of nitroxide radicals by phenolic and thiol antioxidants
Reactions of nitric oxide with phenolic antioxidants and phenoxy radicals
Conclusion
References
9. Tempol: An Ocular Neuroprotectant
10. Miracle Drug Tempol in Cancer Treatment
11. Tempol as Reactive Oxygen Inhibitor
12. Nano-formulations of Tempol
13. Safe Handling, Storage, and Disposal of 4-Hydroxy- TEMPO in Compliance with Pharmaceutical Regulations
Introduction
Physical properties
Storage
Disposal
Safety measures
Precautions
First AID measures
Handling and storage
Toxicological information
First AID measures
Firefighting measures
Accidental release measures
Conclusion
References

Citation preview

Chemical and Clinical Applications of Tempol A comprehensive and authoritative exploration of Tempol (4‑Hydroxy‑ TEMPO), an exceptional chemical compound with diverse ­applications in both scientific research and medical practice. This book delves into Tempol’s unique properties, mechanisms of action, and its potential role in combating ­oxidative stress‑related disorders. It includes a chapter devoted to the safe handling, storage, and disposal of Tempol in compliance with pharmaceutical regulations. The authors pay particular attention to pharmaceutical regula‑ tions in the industry.

Chemical and Clinical Applications of Tempol A Marvelous Molecule

Abhishek Tiwari, Varsha Tiwari and Bimal K. Banik

First edition published 2025 by Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, Florida 33487, U.S.A. and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN CRC Press is an imprint of Taylor & Francis Group, LLC © 2025 Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilm‑ ing, and recording, or in any information storage or retrieval system, without writ‑ ten permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978‑750‑8400. For works that are not avail‑ able on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. ISBN: 9781032730028 (hbk) ISBN: 9781032731223 (pbk) ISBN: 9781003426820 (ebk) DOI: 10.1201/9781003426820 Typeset in Times by codeMantra

Contents Preface vii Author biographies ix Acknowledgments xiii 1 Introduction to Tempol Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

1

2 Synthesis and chemical reactions of Tempol Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

18

3 Tempol in the synthesis of Terpenoids Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

33

4 Name reactions involved in TEMPOL Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

43

5 Industrial applications of TEMPOL 53 Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik 6 The Role of Tempol in NRTI-Induced Mitochondrial toxicity 65 Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik 7 The significance of Tempol in diabetic nephropathy Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

75

v

vi Contents 8 Mechanistic insights into the reaction between a nitroxide radical (Tempol) and a phenolic antioxidant Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik 9 Tempol: an ocular neuroprotectant Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

86

97

10 Miracle drug Tempol in cancer treatment Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

111

11 Tempol as reactive oxygen inhibitor Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

127

12 Nano-formulations of Tempol Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

149

13 Safe handling, storage, and disposal of 4-HydroxyTEMPO in compliance with pharmaceutical regulations Abhishek Tiwari, Varsha Tiwari, and Bimal Krishna Banik

163

Preface In the dynamic world of chemical and clinical research, certain molecules stand out for their remarkable versatility and potential. One such molecule is Tempol, also known as 4‑Hydroxy‑TEMPO, which has garnered signifi‑ cant attention due to its wide‑ranging applications across various scientific and industrial domains. This book, Chemical and Clinical Applications of Tempol: A Marvelous Molecule, aims to provide a comprehensive overview of the multifaceted roles of Tempol, from its fundamental properties and syn‑ thesis to its impactful applications in medicine and industry. Chapter 1, “A Brief Account on Tempol,” introduces the molecule, exploring its history, structure, and the initial discoveries that highlighted its potential. This chapter sets the stage for a deeper understanding of Tempol’s significance in the scientific community. In Chapter 2, “Synthesis and Chemical Reactions of Tempol,” we delve into the methodologies employed to synthesize Tempol and the various chem‑ ical reactions it undergoes. This chapter provides essential knowledge for researchers interested in the practical aspects of working with this molecule. Chapter 3, “Tempol in the Synthesis of Terpenoids,” illustrates how Tempol serves as a valuable reagent in the synthesis of terpenoids, a class of organic compounds with numerous applications in pharmaceuticals and biotechnology. The exploration continues in Chapter 4, “Name Reactions Involved in Tempol,” where we discuss prominently named reactions that utilize Tempol, emphasizing its role in facilitating key chemical transformations. In Chapter 5, “Industrial Applications of Tempol,” we shift our focus to the commercial realm, detailing how Tempol is employed in various indus‑ tries, including its uses in manufacturing processes and product development. Chapter 6, “The Role of Tempol in NRTI‑Induced Mitochondrial Toxicity,” examines the clinical implications of Tempol, particularly its abil‑ ity to mitigate mitochondrial toxicity induced by nucleoside reverse tran‑ scriptase inhibitors (NRTIs), which are crucial in HIV treatment regimens. The therapeutic potential of Tempol is further explored in Chapter 7, “The Significance of Tempol in Diabetic Nephropathy,” where we highlight its protective effects against diabetic kidney disease, a major complication of diabetes. vii

viii Preface In Chapter 8, “Mechanistic Insights into the Reaction Between a Nitroxide Radical (Tempol) and a Phenolic Antioxidant,” we provide a detailed analysis of the interactions between Tempol and phenolic antioxidants, shedding light on the underlying mechanisms that drive these reactions. Chapter 9, “Tempol: An Ocular Neuroprotectant,” focuses on the neuro‑ protective properties of Tempol, particularly in the context of ocular health, offering insights into its potential to prevent or treat neurodegenerative condi‑ tions affecting the eye. The promising role of Tempol in oncology is discussed in Chapter 10, “Miracle Drug Tempol in Cancer Treatment.” This chapter explores the emerging evidence supporting Tempol’s effectiveness as an adjunct in cancer therapy, highlighting its potential to enhance treatment outcomes. Chapter 11, “Tempol as a Reactive Oxygen Inhibitor,” delves into the molecule’s ability to inhibit reactive oxygen species, positioning Tempol as a critical agent in combating oxidative stress‑related conditions. The innovative realm of nanotechnology is covered in Chapter 12, “Nano‑Formulations of Tempol,” where we discuss the development and advantages of nano‑formulations, providing a forward‑looking perspective on Tempol’s application in advanced drug delivery systems. Finally, Chapter 13, “Safe Handling, Storage, and Disposal of 4‑Hydroxy‑TEMPO in Compliance with Pharmaceutical Regulations,” addresses the practical aspects of working with Tempol, ensuring that researchers and industry professionals adhere to best practices and regula‑ tory requirements to maintain safety and efficacy. This book aims to serve as an invaluable resource for chemists, clini‑ cians, and industry professionals alike, offering a thorough understanding of Tempol’s chemical properties, applications, and therapeutic potential. We hope that the insights and knowledge shared within these pages will inspire further research and innovation, harnessing the full potential of this marvel‑ ous molecule.

Author biographies Dr.  Abhishek Tiwari is working as Professor and Head in the Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy. Amity University Lucknow Campus, Lucknow, India.  He obtained his BPharm degree from Jiwaji University Gwalior (MP), MPharm from MS Ramaiah College of Pharmacy, Bengaluru (Karnataka), and PhD from Uttarakhand Technical University, Dehradun (Uttarakhand). He has received grants of more than Rs. 8.5  million from the Department of Biotechnology, Uttarakhand; All India Council of Technical Education, India; CCRUM, India; and Power Finance Corporation. He has been granted 25 granted patents and 11 design patents. In addition, he has submitted a number of patents for approval. He is an editorial mem‑ ber of various national and international journals. He received “Outstanding Academic and Research Award” in 2021, “Best Author Award” in 2019, “Best Teacher Award” in 2019, and “Outstanding Teacher Award” in 2014 and 2015. He has published 78 papers in international journals. He is a rec‑ ognized PhD supervisor of Uttarakhand Technical University, Dehradun and Amity University, Lucknow Campus. He has mentored around 80 students in research, including 2 PhD research scientists. Dr. Varsha Tiwari is working as Professor in the Department of Pharmacognosy, Amity Institute of Pharmacy. Amity University Lucknow Campus, Lucknow, India.  She obtained her BPharm degree from Jiwaji University Gwalior (MP), MPharm. from MS Ramaiah College of Pharmacy, Bengaluru (Karnataka), and PhD from Uttarakhand Technical University, Dehradun (Uttarakhand). She has been granted 24 patents and 11 design patents. In addi‑ tion, she has submitted a number of patents for approval. She has received “Young scientist Award” in 2022, “Young Teacher Award” in 2020, “Pharma ix

x  Author biographies Recognition Award” in 2019, and “Outstanding Teacher Award” in 2015. She has published 68 research papers in national and international journals. Remarkably, she has published more than 15 books and 20 chapters. She is a recognized PhD supervisor of Amity University, Lucknow Campus, India. She has delivered several lectures as an eminent speaker in National and International Conferences. Her core area includes diabetes, nanotechnol‑ ogy, phytochemistry, and chromatography‑based analysis. She has mentored around 88 students in research, including 1 PhD research scientists. Prof. Bimal Krishna Banik conducted his doctoral research at the Indian Association for the Cultivation of Science, Calcutta. Then, he pursued postdoctoral research at Case Western Reserve University and Stevens Institute of Technology. He was a Tenured Full Professor of Chemistry and First President’s Endowed Professor of Science & Engineering at the University of Texas‑Pan American. He was also the Vice President of Research & Education Development of the Community Health Systems of Texas. At present, he is a full professor of the Deanship of Research Development at the Prince Mohammad Bin Fahd University (Kingdom of Saudi Arabia). Professor Banik taught chemistry to BS, MS, and PhD students in the United States and Saudi Arabia universities for many years. In research, he directly mentored approximately 300 students, 20 postdoctoral fellows, 7 PhD research scientists, and 28 university/college faculties. He acted as the advi‑ sor of two students’ organizations that had 1,400 students. As the principal investigator (PI), he was awarded $7.25 million in grants from NIH and NCI. Importantly, he has about 680 publications along with more than 500 presen‑ tation abstracts. Many of his international presentations were designated as Keynote and Plenary lectures. Professor Banik served as the PI of a joint green chemistry symposium between the United States and India. He chaired 20 symposiums at the American Chemical Society (ACS) National Meetings and over two dozen at the International level, including one at the Nobel Prize Celebration. In the capacity of chair, he introduced about 300 speakers. He is a reviewer of 93, editorial board member of 26, editor‑in‑chief of 12, founder of 8, and guest editor of 10 research journals. As the editor‑in‑chief, he recruited approxi‑ mately 200 associate editors and editorial board members. He is an examiner

Author biographies  xi of NSF, NCI, NIH, NRC, DOE, ACS, and International grant applications; and a panel member of NSF and NCI/NIH grant sections. He served as the chair/member of more than 100 scientific committees. The number of cita‑ tions for his publications is more than 10,000. Professor Banik was given the Indian Chemical Society’s Life‑Time Achievement Award; Mahatma Gandhi Pravasi Honor Medal from the UK Parliament; US National Society of Collegiate Scholars’ Best Advisor Award for students; Professor P. K. Bose Medal; Dr.  M. N. Ghosh Gold Medal; University of Texas Board of Regents’ Outstanding Teaching Award; and ACS Member Service Award.

Acknowledgments This handbook will not be published without the assistance of CRC, Taylor & Francis Publisher. In particular, the authors are tremendously grateful to Ms. Hilary Lafoe and Varalika Kathuria for their knowledge in science, helping attitude, and encouragement.

xiii

Introduction to Tempol

1

Abhishek Tiwari1*, Varsha Tiwari2*, and Bimal Krishna Banik3*

INTRODUCTION Nitroxyl radicals, commonly known as nitroxides, are characterized as N, N‑disubstituted NO radicals wherein an unpaired electron is delocalized between the nitrogen and oxygen atoms. This delocalization is exemplified by two resonance structures, indicating the distribution of spin density between both atoms, often with a slightly higher density at the oxygen atom. The earli‑ est synthesis of an inorganic nitroxyl radical dates back to 1845 when potas‑ sium nitroso disulfonate was prepared by Fremy. Later, in 1901, Piloty and Schwerin achieved the synthesis and isolation of porphyrexide 4, marking the first instance of an organic nitroxide. Subsequent work by Offenbächer and Wieland led to the preparation and isolation of diphenylnitroxide, a compound previously believed to be unstable. Among the array of nitrox‑ ides, 2,2,6,6‑tetramethylpiperidine‑N‑oxyl radical (TEMPO) stands out as a prominent member. First synthesized by Lebedev and Kazarnovskii in 1959, Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 2 Department of Pharmacognosy, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 3 Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University, Al Khobar 31952, Kingdom of Saudi Arabia; 1

*

Corresponding Authors: [email protected]; [email protected]; [email protected]

DOI: 10.1201/9781003426820-1

1

2  Chemical and Clinical Applications of Tempol TEMPO and many other nitroxides exhibit stability at room temperature, falling under the category of persistent radicals. The delocalization energy for the unpaired electron in these compounds is estimated to be around 120 kJ/mol, attributed to the presence of a three‑electron N–O bond. The kinetic stability of dialkyl nitroxides is bolstered by the steric hindrance imposed by their substituents, shielding the nitroxide functionality. However, nitrox‑ ides with heteroatom or aryl substituents tend to be less stable compared to alkyl‑substituted nitroxides. In diaryl‑substituted nitroxides, the delocaliza‑ tion of the radical into the aromatic ring weakens the nitrogen–oxygen bond [1, 2]. The selected nitroxide radicals are shown in Figure 1.1 (Figure 1). Tempol, chemically represented as C9H18NO2, belongs to the class of stable free radicals known as nitroxides. TEMPO is a member of the class of aminoxyls that is piperidine that carries an oxidanediyl group at posi‑ tion 1 and methyl groups at positions 2, 2, 6, and 6, respectively (Figure 1.1; Figure 2). The piperidine ring serves as the backbone of Tempol’s structure, providing stability to the nitroxide radical while allowing for interactions with other molecules. The presence of the hydroxyl group enhances Tempol’s solubility and reactivity in aqueous environments, contributing to its efficacy as an antioxidant and its potential therapeutic applications. It has a role as a ferroptosis inhibitor, a catalyst and a radical scavenger. It is a member of piperidines and a member of aminoxyls. Originally synthesized for its role as a spin label in electron paramagnetic resonance (EPR) ­spectroscopy, Tempol has since garnered attention for its antioxidant properties, making it a sub‑ ject of interest in fields ranging from chemistry to medicine. TEMPO finds applications in organic chemistry as a radical trap, catalyst, and mediator in polymerization processes. It is utilized as a spin label in magnetic resonance imaging (MRI). Additionally, it has applications in various fields, such as electrochemistry, sensors, and medicinal chemistry [1–3]. The X‑ray crystallographic analysis of TEMPO and its corresponding oxoammonium cation provides valuable insights into their structural charac‑ teristics upon oxidation. These investigations reveal that upon oxidation, both TEMPO and its oxoammonium cation undergo relatively minor structural alterations. Specifically, there is a slight reduction in the N–O bond length TABLE 1.1  X‑ray properties of nitroxide, oxoammonium cation, and amine

ELECTRONIC STATE N ATOM N–O bond length (Å) C–N–O bond angle (°) Deviation from C2O Plane (Å)

NITROXIDE

OXOAMMONIUM CATION

AMINE

SP3/SP2

SP2

SP3

1.28 112 0.177

1.18 122 0.007

– 106.7 –

1  •  Introduction to Tempol  3 by 0.1 Å, accompanied by a marginal deviation of the nitrogen atom from the C2O plane. These subtle changes are crucial as they contribute signifi‑ cantly to the high redox stability, often termed as cyclability, exhibited by the nitroxide/oxoammonium (NO•/N+=O) redox couple. Figure  1.1 (Figure  3) and Table 1.1 illustrate these structural transformations, showcasing the intri‑ cate molecular arrangements of TEMPO and its oxoammonium counterpart. Through this detailed analysis, we gain a deeper understanding of the mecha‑ nisms underlying the rapid electron transfer processes characteristic of the NO•/N+=O redox couple [4, 5]. Tempol was first synthesized and characterized in the 1950s by organic chemists exploring stable free radicals. The synthesis of Tempol involved the oxidation of the corresponding amine precursor, resulting in the formation of the stable nitroxide radical [6]. In the 1970s and 1980s, researchers began to recognize Tempol’s potent antioxidant properties. Studies demonstrated its ability to scavenge reactive oxygen species (ROS) and protect against oxida‑ tive damage [7]. TEMPO is a dark red solid at room temperature, with a melt‑ ing point ranging from 32°C to 37°C. As for the smell, tempo is described as odorless. Generally, Tempol is soluble in polar solvents such as water, ethanol, methanol, and acetone. It is sparingly soluble in non‑polar solvents like hexane and chloroform. The specific density of tempo is approximately 1.08 g/cm³ at 20°C. The specific rotation of TEMPO is +136° (c=1, methanol). TEMPO is classified as corrosive and irritant, capable of causing severe skin burns and eye damage. It may also cause respiratory irritation and is harm‑ ful to aquatic life with long‑lasting effects. Acute toxicity studies suggest that it can induce somnolence and skin burns in animal models. Over time, Tempol’s applications expanded beyond spectroscopy to include radiopro‑ tection, neuroprotection, and cardioprotection. Its versatility and efficacy in various fields made it a subject of extensive research [8]. Hydroxylamines exhibit remarkably weak O–H bonds, with energy val‑ ues of 69.6 and 71.8 kcal/mol for hydroxylamines derived from TEMPO and TEMPONE, respectively. Consequently, nitroxides do not engage in hydro‑ gen abstraction reactions due to this weakness. Nevertheless, owing to their radical nature, nitroxides readily react with other radicals, thereby serving as antioxidants. These compounds can swiftly interact with various biologi‑ cally relevant radical oxidants and reductants, while undergoing recycling through the formation of oxoammonium cations and hydroxylamine deriva‑ tives (Figure  1.1; Scheme 1). Nitroxides may be depleted when they react with biological molecules or certain radicals, leading to the generation of the corresponding amine, as observed with thiyl radicals, or when the oxoam‑ monium cation is notably unstable, as seen in the case of TEMPONE and 4‑amino‑TEMPO. The highly oxidizing oxoammonium cation exhibits the capacity to selectively oxidize primary alcohols present in mono‑ and poly‑ saccharides, a process catalyzed by nitroxides (Scheme 2). Additionally, the

4  Chemical and Clinical Applications of Tempol formation of the oxoammonium cation could contribute to the pro‑oxidative activity and potential adverse effects of nitroxides, which otherwise function as antioxidants (Figure 1.1; Scheme 3) [9–16].

MECHANISMS OF NITROXIDE REACTIONS WITH BIOLOGICALLY RELEVANT SMALL RADICALS The reactions of radicals such as •NO2, CO3•−, HO2•, and RO2• (referred to as X•) with nitroxides result in the formation of a common intermediate. This intermediate has the capability to oxidize compounds like ferrocyanide, NADH, and 2,2‑azino‑bis(3‑ethylbenzothiazoline‑6‑sulfonate) (ABTS2−). In certain instances, spectroscopic analysis identified the intermediate as the oxoammonium cation. Kinetic studies indicate that the generation of the oxoammonium cation follows an inner sphere electron transfer mechanism, as illustrated in Schemes 2 and 3 [9–16]. This conclusion was reached by applying Marcus theory of oxidation‑ reduction reactions to elucidate the formation of the oxoammonium cation as depicted in Figure 1.1 (Scheme 3) [9–16].

APPLICATIONS OF TEMPOL Pavithra et al. reported the synthesis of xanthenediones and acridinediones utilizing TEMPO/CuCl2  — catalyzed one‑pot aerobic oxidation represents a significant advancement in organic synthesis, offering several notable advan‑ tages (Figure 1.1; Scheme 4). The accessibility of the required reagents and catalysts further enhances the attractiveness of this method. TEMPO and CuCl2, the catalysts involved in the reaction, are generally readily avail‑ able in most laboratory settings. This accessibility contributes to the wide‑ spread adoption of the synthesis protocol. Furthermore, the use of one‑pot aerobic oxidation eliminates the need for multiple reaction steps or additional reagents, simplifying the synthetic pathway. This streamlined approach reduces the overall reaction complexity and minimizes the risk of side reac‑ tions or by‑product formation [17]. In their research published in Organic Letters Cai et al., developed a novel method for the synthesis of 2‑acylpyrroles, addressing several key challenges

1  •  Introduction to Tempol  5 associated with traditional synthetic approaches (Figure 1.1; Scheme 5). The researchers introduced a groundbreaking organotin‑free methodology, which represents a significant departure from conventional strategies that often rely on organotin compounds. By eliminating organotin reagents from the syn‑ thetic pathway, the team aimed to enhance the safety and sustainability of the synthesis process. The method developed by Cai et al. does not require the use of initiators, simplifying the reaction conditions and reducing the risk of side reactions or unwanted by‑products. This omission of initiators streamlines the synthetic protocol, making it more accessible and efficient. Also, the research‑ ers conducted the synthesis under mild conditions, avoiding harsh reaction parameters that can lead to decreased selectivity and yields. The use of mild conditions enhances the overall efficiency of the reaction while ensuring the safety and ease of handling in the laboratory. This developed methodology circumvents the use of toxic or hazardous reagents such as azo compounds and peroxides, aligning with principles of green chemistry and promoting safer laboratory practices. This eco‑friendly approach reduces environmental impact and minimizes health risks associated with hazardous substances [18]. Zhang et  al. reported an innovative synthetic approach for the prepa‑ ration of 2,5‑disubstituted 1,3,4‑oxadiazole compounds, addressing several important considerations inherent in traditional synthetic methodologies (Figure  1.1; Scheme 6). One of the key highlights of their research is the broad scope of their synthetic method, allowing for the efficient preparation of a wide range of 2,5‑disubstituted 1,3,4‑oxadiazole derivatives. This broad scope provides researchers with a versatile platform for accessing diverse molecular structures, facilitating the exploration of structure‑activity relation‑ ships and potential applications in various fields. Additionally, the synthetic protocol exhibits good functional group tolerance, enabling the incorpora‑ tion of different chemical functionalities into the final product without com‑ promising reaction efficiency or selectivity. This aspect of the methodology enhances its utility in complex molecule synthesis, where the presence of multiple functional groups can pose challenges to traditional synthetic approaches. Another significant advantage of the developed methodology is the high yields achieved under mild reaction conditions in the presence of oxygen (O2). The use of mild conditions minimizes the risk of side reactions or unwanted by‑products while ensuring optimal efficiency and reproducibil‑ ity of the synthesis. Furthermore, the utilization of O2 as a reagent enhances the sustainability of the synthetic process, aligning with principles of green chemistry by reducing the reliance on environmentally harmful reagents [19]. Chu et  al. reported an innovative synthetic strategy for the preparation of pyrimidine derivatives, addressing several key challenges associated with traditional synthetic methodologies (Figure 1.1; Scheme 7). One of the note‑ worthy aspects of their research is the utilization of a recyclable iron cata‑ lyst generated in situ. Transition metal catalysts play a crucial role in many

6  Chemical and Clinical Applications of Tempol organic transformations, but their efficient recovery and reuse are often hin‑ dered by practical challenges. By employing a catalyst that can be generated in situ and recycled, the researchers have developed a more sustainable and cost‑effective synthetic protocol. Furthermore, the synthetic method enables the β‑functionalization of saturated carbonyl compounds, allowing for the introduction of diverse functional groups at the β‑position relative to the car‑ bonyl moiety. This functional group tolerance enhances the versatility of the synthetic approach, enabling the preparation of a wide range of pyrimidine derivatives with tailored chemical properties. Additionally, the synthetic proto‑ col involves the cleavage of three C–H bonds and three N–H bonds, facilitating the construction of the pyrimidine ring system from readily available starting materials. This step‑efficient approach streamlines the synthetic pathway and minimizes the number of synthetic steps required for the preparation of pyrimi‑ dine derivatives, enhancing the overall efficiency of the synthesis [20]. Zhang et  al. investigated a groundbreaking synthetic approach for the construction of pyrimidine scaffolds via unactivated β‑C(sp3)–H function‑ alization of saturated ketones, addressing several key challenges associated with traditional synthetic methodologies (Figure 1.1; Scheme 8). A notable highlight of their research is the pioneering demonstration of the first example for the construction of pyrimidine scaffolds through unactivated β‑C(sp3)–H functionalization of saturated ketones. Traditional methods for pyrimi‑ dine synthesis often involve multiple synthetic steps and functional group manipulations, making the synthesis challenging and resource‑intensive. By harnessing the reactivity of unactivated β‑C(sp3)–H bonds, the research‑ ers have developed a more direct and efficient route to pyrimidine deriva‑ tives. Moreover, the synthetic pathway proceeds through a radical pathway, offering a versatile and atom‑economic approach to pyrimidine synthesis. Radical‑based transformations enable the rapid generation of molecular com‑ plexity from simple starting materials, facilitating the construction of diverse molecular architectures with high efficiency and selectivity. Furthermore, the synthetic protocol is designed as a one‑pot strategy, allowing for the sequen‑ tial execution of multiple synthetic steps in a single reaction vessel. This streamlined approach minimizes the number of purification steps and inter‑ mediate handling, reducing the overall synthetic effort and increasing the efficiency of the synthesis. Additionally, the developed methodology exhibits good functional group tolerance, enabling the incorporation of various chem‑ ical functionalities into the final product without compromising reaction effi‑ ciency or selectivity. This aspect enhances the versatility and applicability of the synthetic approach, allowing for the preparation of structurally diverse pyrimidine derivatives tailored for specific applications [21]. Chen et  al. published a novel and efficient method for synthesizing acylpyridines and pyridine‑3‑carboxylates was introduced (Figure  1.1; 3‑­ Scheme 9). The method involves oxidative one‑pot sequential reactions of

1  •  Introduction to Tempol  7 inactivated saturated ketones with electron‑deficient enamines. The research presents a cascade C(sp3)–H functionalization strategy for the synthesis of pyridines, wherein multiple C–H bonds are sequentially functionalized in a single synthetic step. This approach streamlines the synthetic pathway, allow‑ ing for the rapid construction of complex pyridine structures from readily available starting materials. One notable aspect of this synthetic methodology is its broad substrate scope, enabling the synthesis of a wide variety of pyri‑ dine derivatives. This versatility enhances the applicability of the method, facilitating the preparation of diverse compounds with tailored chemical properties for various applications in organic synthesis and medicinal chem‑ istry. Moreover, the reaction conditions employed in this synthetic protocol are simple, requiring readily available reagents and operating under mild conditions. This feature enhances the accessibility of the method, making it suitable for use in both academic and industrial laboratories. Furthermore, the synthetic approach exhibits excellent regioselectivity, ensuring the selec‑ tive functionalization of specific C–H bonds within the substrate molecules. This high regiocontrol is crucial for the efficient synthesis of target com‑ pounds, minimizing the formation of unwanted by‑products and simplifying the purification process [22]. Xu et  al. developed an innovative method for the synthesis of ben‑ zothiazoles, addressing several key challenges associated with traditional synthetic methodologies (Figure  1.1; Scheme 10). A notable highlight of their research is the development of a transition‑metal‑free synthetic approach for benzothiazole synthesis. Transition metals are commonly used as catalysts in organic synthesis, but they can be expensive and envi‑ ronmentally hazardous. By eliminating the need for transition metals, the researchers have devised a more sustainable and cost‑effective synthetic route. Moreover, the synthetic method is photosensitizer‑free, avoiding the use of light‑sensitive compounds that can complicate the reaction setup and increase the risk of side reactions. This aspect enhances the reliability and reproducibility of the synthetic procedure, making it more practical for large‑scale applications. Additionally, the synthetic protocol is base‑free, eliminating the requirement for basic reagents that can be corrosive and difficult to handle. This simplifies the reaction conditions and reduces the need for extensive purification steps, enhancing the overall efficiency of the synthesis. Furthermore, the synthetic approach is compatible with a wide range of functional groups, allowing for the incorporation of diverse chemical functionalities into the final product. This versatility enables the preparation of structurally diverse benzothiazole derivatives tailored for specific applications in organic synthesis, medicinal chemistry, and mate‑ rials science [23]. Lee, J. W. 2020 reported the first‑ever application of redox‑neutral TEMPO• catalysis for achieving intermolecular di‑ and trifluoromethoxylation

8  Chemical and Clinical Applications of Tempol of (hetero)arenes (Figure 1.1; Scheme 11). This innovative method represents a significant advancement in the field of fluorination chemistry, offering sev‑ eral key advantages over traditional approaches. One of the primary high‑ lights of their work is the utilization of TEMPO• as a redox‑neutral catalyst for di‑ and trifluoromethoxylation reactions. This approach circumvents the need for transition metal catalysts, which are often expensive and environ‑ mentally unfriendly. By harnessing the redox properties of TEMPO•, the researchers were able to facilitate the fluorination process under mild reac‑ tion conditions without the generation of toxic or hazardous by‑products. Another notable aspect of their methodology is the use of readily available and inexpensive TEMPO• catalyst. Unlike many transition metal catalysts, which can be prohibitively expensive and difficult to access, TEMPO• offers a cost‑effective alternative that is readily available in the laboratory. This accessibility contributes to the practicality and scalability of the di‑ and trifluoromethoxylation process, making it more widely applicable to both academic and industrial settings. Furthermore, the developed protocol dem‑ onstrates exceptional functional group tolerance, enabling the fluorination of (hetero)arenes containing a diverse array of functional groups. This broad compatibility expands the scope of substrates amenable to di‑ and trifluoro‑ methoxylation, allowing for the synthesis of complex molecules with multiple functional handles [24]. Zhang et al. reported a novel biomimetic aerobic oxidation method for the conversion of alcohols to carbonyl compounds (Figure 1.1; Scheme 12). This innovative approach harnesses the synergistic effects of a mixture com‑ posed of FeCl3, L‑valine, and TEMPO to facilitate the oxidation process under mild reaction conditions. The developed methodology demonstrates excellent efficiency in the oxidation of alcohols to carbonyl compounds, achieving good to exceptional isolated yields. This high yield is indicative of the effectiveness and reliability of the oxidation protocol. One of the key advantages of this biomimetic oxidation method is its remarkable compat‑ ibility with various functional groups present in the substrate molecules. The oxidation process proceeds smoothly without compromising the integrity of sensitive functional groups, thereby allowing for the synthesis of diverse car‑ bonyl compounds. The use of mild reaction conditions is a significant advan‑ tage of this oxidation protocol. By operating under mild conditions, such as ambient temperature and atmospheric pressure, the researchers minimize the need for harsh reaction conditions, thereby enhancing the safety and sustain‑ ability of the process [25]. Zhang et al. reported a novel iron‑catalyzed 1,2‑dehydrogenation method for the transformation of carbonyl compounds into their α, β‑unsaturated equivalents (Figure 1.1; Scheme 13). The use of iron as a catalyst represents a significant advancement in catalytic methodology. Base‑metal catalysis

1  •  Introduction to Tempol  9 offers several advantages over traditional transition‑metal catalysts, includ‑ ing cost‑effectiveness and environmental friendliness. By employing iron as the catalyst, the researchers enhance the sustainability and accessibility of the dehydrogenation process. The developed methodology demonstrates a broad substrate scope, accommodating various carbonyl compounds and analogues. This includes aldehydes, ketones, lactones, lactams, amines, and alcohols,  highlighting the versatility and applicability of the dehydro‑ genation protocol. The ability to transform diverse substrates into their α, β‑unsaturated counterparts underscores the utility of this methodology in organic synthesis. One of the key advantages of this dehydrogenation method is its simplicity and efficiency. The transformation occurs in a single step, facilitating the rapid generation of α, β‑unsaturated carbonyl compounds with good yields. This streamlined reaction protocol simplifies the synthetic route and minimizes the number of synthetic steps required, thereby improv‑ ing overall synthetic efficiency [26]. Jiang et  al. developed a sustainable oxidation technology for the con‑ version of alcohols (aldehydes) to carboxylic acids (Figure 1.1; Scheme 14). The oxidation process utilizes O2 or air as the terminal oxidant, eliminating the need for more hazardous or expensive oxidizing agents. This choice of oxidant contributes to the sustainability and eco‑friendliness of the oxida‑ tion technology, aligning with principles of green chemistry. The use of a catalytic amount of Fe(NO3)3·9H2O/TEMPO/KCl enables the efficient con‑ version of alcohols (aldehydes) to carboxylic acids in high yields. This high efficiency underscores the practical utility of the developed methodology for synthetic applications. Importantly, the oxidation reactions proceed smoothly at ambient temperature, demonstrating the mild reaction conditions afforded by the catalytic system. The ability to carry out the oxidation process at room temperature enhances the practicality and accessibility of the method‑ ology, reducing the need for energy‑intensive heating or cooling processes. The scalability of the oxidation technology further enhances its applicability in synthetic chemistry. The demonstrated effectiveness of the method on a larger scale indicates its potential for industrial applications and the synthesis of carboxylic acids in bulk quantities [27]. Furukawa et al. reported a significant advancement in the catalytic oxida‑ tion of 1,2‑diols to α‑hydroxy acids (Figure 1.2; Scheme 15). The developed catalytic system enables the selective oxidation of 1,2‑diols to α‑hydroxy acids. This chemo‑selectivity is crucial for controlling the reaction pathway and avoiding undesired oxidative cleavage or overoxidation products, leading to improved synthetic efficiency. The oxidation process involves the forma‑ tion of a charge‑transfer complex facilitated by the catalytic system composed of TEMPO, NaOCl, and NaClO2. This complexation likely plays a crucial role in directing the selective oxidation of 1,2‑diols to α‑hydroxy acids while

10  Chemical and Clinical Applications of Tempol FIGURE 1.1  Structures of selected nitroxide radicals; Figure 2: (a) 2D‑structure of Tempol, (b) 3D‑‑structure of Tempol; Figure 3: X‑ray crystal structures of nitroxide, oxoammonium cation, and amine; Scheme 1: One‑electron oxidation and reduction processes converting nitroxide to oxoammonium cation and hydroxylamine, respectively; Scheme 2: Schematic representation illustrating the inner sphere electron transfer mechanism leading to the formation of the oxoammonium cation from the reactions of radicals (X•) with nitroxides, and subsequent oxidation; Scheme 3: Application of Marcus theory of oxidation‑reduction reactions to elucidate the formation of the oxoammonium cation; Scheme 4: Synthesis of xanthenediones and acridinediones; Scheme 5: Synthesis of 2‑acylpyrroles; Scheme 6: Synthesis of 2,5‑disubstituted 1,3,4‑oxadiazole; Scheme 7: Synthesis of pyrimidines; Scheme 8: Synthesis of pyrimidines; Scheme 9: Synthesis of pyridines; Scheme 10: Synthesis of benzothiazoles; Scheme 11: Di‑ and trifluoromethoxylation; Scheme 12: Biomimetic aerobic oxidation of alcohols; Scheme 13: α,β‑Dehydrogenation; Scheme 14: Oxidation from alcohols (also aldehydes) to carboxylic acids

1  •  Introduction to Tempol  11 minimizing side reactions. The reported methodology allows for the synthe‑ sis of optically active α‑hydroxy acids, highlighting its potential utility in asymmetric synthesis and the preparation of chiral molecules. This aspect expands the scope of the catalytic oxidation process to access a diverse range of enantiomerically enriched α‑hydroxy acids. The use of a two‑phase system comprising hydrophobic toluene and water facilitates the oxidation reaction by reducing the accompanying oxidative cleavage. This setup enhances the efficiency and selectivity of the oxidation process, contributing to improved yields of the desired α‑hydroxy acids [28]. Noh and Kim reported a novel method for the synthesis of nitriles from aldehydes utilizing a nitroxyl radical/NOx system under aerobic conditions (Figure  1.2; Scheme 16). The reported approach represents a significant advancement as it does not rely on transition metals for catalyzing the conver‑ sion of aldehydes to nitriles. This transition metal‑free catalytic system offers advantages in terms of cost‑effectiveness, environmental friendliness, and potentially avoiding issues related to metal contamination in the final prod‑ uct. The oxidation process occurs under aerobic conditions, which means that molecular oxygen (O2) serves as the terminal oxidant. This feature enhances the sustainability of the process by utilizing readily available and environ‑ mentally benign oxygen as the oxidizing agent, eliminating the need for addi‑ tional chemical oxidants. The developed methodology demonstrates a broad substrate scope, allowing for the conversion of various aldehydes to their cor‑ responding nitriles. This broad applicability enhances the versatility and util‑ ity of the approach for the synthesis of diverse nitrile compounds, potentially useful in pharmaceuticals, agrochemicals, and other industrial applications. Additionally, the study showcases a one‑pot sequential approach, enabling the direct conversion of primary alcohols to nitriles via aerobic oxidation without the need for intermediate isolation. This strategy streamlines the syn‑ thetic process, reduces the number of synthetic steps, and improves overall efficiency [29]. Zhang et al. reported an innovative method for the synthesis of N‑sulfinyl and N‑sulfonylimines via the oxidation of alcohols followed by condensation with sulfinamide or sulfonamide, all achieved in a single pot under benign conditions (Figure 1.2; Scheme 17). The study presents the utilization of an Fe(III) catalyst in conjunction with L‑valine and 4‑OH‑TEMPO to facili‑ tate the oxidation of alcohols and subsequent condensation with sulfinamide or sulfonamide. The use of iron as the catalyst offers several advantages, including abundance, low cost, and reduced environmental impact com‑ pared to transition metal catalysts. The reported method represents the first example of an Fe‑catalyzed aerobic oxidative one‑pot synthesis of N‑sulfinyl and N‑sulfonylimines directly from alcohols. This streamlined synthetic approach eliminates the need for multiple reaction steps and intermediate

12  Chemical and Clinical Applications of Tempol isolation, thereby enhancing overall efficiency and atom economy. The developed catalytic system exhibits high tolerance toward various func‑ tional groups present in the substrate molecules. This broad functional group compatibility expands the scope of accessible substrates and allows for the synthesis of diverse N‑sulfinyl and N‑sulfonylimine compounds with varied chemical functionalities. Importantly, the oxidation of alcohols and subse‑ quent condensation steps are conducted under benign reaction conditions. The use of mild conditions contributes to the practicality and applicability of the methodology while minimizing the generation of waste and reducing energy consumption [30]. Chamorro‑Arenas et al. reported a novel and environmentally friendly protocol for the selective and catalytic TEMPO C(sp3)–H oxidation of piperazine and morpholines (Figure  1.2; Scheme 18). The study presents an unprecedented tandem catalytic fashion for the selective oxidation of C(sp3)–H bonds in piperazine and morpholines. This dual oxidation process enables the conversion of piperazine to 2,3‑diketopiperazines (2,3‑DKP) and morpholines to 3‑morpholinones (3‑MPs) in a single reaction step. The developed protocol utilizes inexpensive and safe reagents, including NaClO2, NaOCl, and catalytic amounts of TEMPO. These reagents are environmen‑ tally friendly and readily available, contributing to the sustainability of the synthetic process. The methodology offers selective and catalytic C(sp3)–H oxidation, allowing for the targeted functionalization of piperazine and mor‑ pholine substrates. The controlled oxidation of specific C–H bonds in the presence of other functional groups demonstrates the high chemo‑selectivity of the developed protocol [31]. The work by Liu et al., reported a metal‑free oxidative dearomatization strategy for indoles using TEMPO oxoammonium salt (Figure 1.2; Scheme 19). This innovative approach enables the transformation of indoles with aro‑ matic ketones, resulting in the formation of 2‑alkoxyamino‑3‑morpholinone derivatives. The methodology offers a broad substrate scope, accommodat‑ ing various indole and ketone derivatives, and exhibits excellent functional group tolerance. By providing a straightforward and efficient route for the synthesis of 2‑alkoxyamino‑3‑morpholinones, this work expands the synthe‑ sis for accessing valuable heterocyclic compounds with potential applications in drug discovery and materials science [32]. Zhang et  al. reported a metal‑free recyclable catalyst system for the selective aerobic oxidation of structurally varied benzylic C(sp3)–H bonds of ethers and alkylarenes (Scheme 20). The study presents a novel catalyst system that is completely metal‑free and recyclable. This system is designed to facilitate the selective aerobic oxidation of benzylic C(sp3)–H bonds in ethers and alkylarenes without the need for transition metal catalysts, which are often costly and environmentally unfriendly. The catalyst system enables

1  •  Introduction to Tempol  13 the selective oxidation of benzylic C(sp3)–H bonds, demonstrating high regi‑ oselectivity in the presence of other reactive sites. This selectivity is cru‑ cial for controlling the reaction outcome and obtaining the desired products with high efficiency. The oxidation reactions proceed under mild conditions, which is advantageous for preserving sensitive functional groups and mini‑ mizing unwanted side reactions. The mild reaction conditions contribute to the overall efficiency and practicality of the methodology. The catalyst sys‑ tem exhibits a broad substrate scope, allowing for the oxidation of structur‑ ally varied benzylic C(sp3)–H bonds present in ethers and alkylarenes. This versatility enables the synthesis of diverse products, including isochroma‑ nones and xanthones, from readily available alkyl aromatic precursors. The oxidation reactions occur under aerobic conditions, utilizing oxygen from the air as the terminal oxidant. This environmentally benign aspect of the meth‑ odology eliminates the need for additional oxidants and reduces the environ‑ mental impact of the process [33]. Xie and Stahl reported on Cu/nitroxyl catalysts for the selective aero‑ bic oxidative lactonization of diols. The catalyst system supports mild reac‑ tion conditions, enabling the oxidative lactonization of diols under ambient temperature with excellent efficiency (Figure 1.2; Scheme 21). The catalyst system demonstrates exceptional chemo‑ and regioselectivity, particularly for the oxidation of less hindered unsymmetrical diols. This selectivity allows for the controlled formation of lactones from diol substrates. The methodology tolerates diverse functional groups, providing flexibility for the synthesis of lactones from diol substrates containing various func‑ tional moieties. This feature enhances the applicability and versatility of the reaction in organic synthesis. Ambient air serves as the oxidant in the reaction, eliminating the need for additional oxidants or harsh reaction conditions. This environmentally benign aspect of the methodology con‑ tributes to its sustainability and practicality. By altering the identity of the nitroxyl cocatalyst, such as switching between TEMPO and ABNO, the chemo‑ and regioselectivity of the reaction can be adjusted. This capability allows for fine‑tuning of the reaction conditions to suit different types of diol ­substrates [34]. Wu et  al. described a novel approach for the catalytic acceptorless dehydrogenation (CAD) of N‑heterocycles, leveraging TEMPO as the organo‑electrocatalyst (Figure 1.2; Scheme 22). The study utilizes TEMPO as the organo‑electrocatalyst, showcasing its effectiveness in facilitating the acceptorless dehydrogenation of N‑heterocycles under electrochemical con‑ ditions. This highlights the versatility of TEMPO as a catalyst in electro‑ chemical transformations. Lei et al. developed a mild and metal‑free route for the dehydrogenation of N‑heterocycles using the CAD strategy. This approach offers advantages over traditional methods by avoiding the use of

14  Chemical and Clinical Applications of Tempol harsh conditions or transition metal catalysts, thus enhancing the sustain‑ ability and efficiency of the process. The CAD strategy demonstrated a broad substrate scope, allowing for the synthesis of a variety of five‑ and six‑­ membered nitrogen‑heteroarenes with high yields. This substrate generality indicates the versatility and applicability of the methodology in the synthesis of diverse N‑heterocyclic compounds [35]. Lu and Shen developed a highly efficient method for synthesizing ­alkenylboronates through copper catalysis (Figure 1.2; Scheme 23). The Cu/ TEMPO catalytic system enables the direct functionalization of both aro‑ matic and aliphatic terminal alkenes. This methodology provides a straight‑ forward route for the transformation of readily available alkenes into valuable alkenylboronate compounds. The catalytic system demonstrates high reactiv‑ ity and selectivity, allowing for the efficient conversion of alkenes and pinacol diboron into alkenylboronates. This high selectivity ensures the formation of the desired products with minimal side reactions, enhancing the efficiency of the synthetic process. The Cu/TEMPO catalytic system exhibits excel‑ lent chemo‑selectivity, regio‑selectivity, and stereoselectivity. This level of selectivity ensures the precise control over the functionalization of alkenes, leading to the formation of alkenylboronates with desired stereochemical and regiochemical properties [36].

CONCLUSION The chapter emphasizes the wide‑ranging applications and significant advancements in the use of TEMPO (2,2,6,6‑tetramethylpiperidin‑1‑yl)oxyl or (2,2,6,6‑tetramethylpiperidin‑1‑yl)oxidanyl) across multiple disciplines. Through its stability and versatility, TEMPO has become a valuable tool in organic synthesis, catalysis, material science, and biological research. The extensive discussion highlights TEMPO’s role as a catalyst, mediator, and radical trap in various synthetic transformations, including the synthesis of diverse heterocyclic compounds, oxidative transformations, and selective oxidation reactions. Notably, TEMPO‑mediated reactions demonstrate broad substrate scope, excellent functional group tolerance, and compatibility with mild reaction conditions, making them highly valuable in synthetic chemis‑ try. TEMPO’s importance in advancing green chemistry practices, as many TEMPO‑catalyzed reactions operate under environmentally friendly condi‑ tions, such as using benign reagents, avoiding toxic by‑products, and employ‑ ing sustainable oxidation technologies.

1  •  Introduction to Tempol  15

FIGURE 1.2  Scheme 15: Synthesis of N‑sulfinyl and N‑sulfonylimines; Scheme 16: Aldehyde to nitrile; Scheme 17: Synthesis of N‑sulfinyl and N‑sulfonylimines; Scheme 18: Dual C(sp3)–H oxidation; Scheme 19: Oxidative dearomatization; Scheme 20: Benzylic oxidation; Scheme 21: Oxidative lactonization of diols; Scheme 22: Catalytic acceptorless dehydrogenation (CAD); Scheme 23: Dehydrogenative borylation

16  Chemical and Clinical Applications of Tempol

REFERENCES 1. Vogler V, Studer A. Applications of TEMPO in Synthesis. Synthesis 2008, 2008 (13), 1979–1993. https://doi.org/10.1055/s‑2008‑1078445 2. Ciriminna R, Pagliaro M. Industrial Oxidations with Organocatalyst TEMPO and Its Derivatives. Org. Process Res. Dev. 2010, 14 (1), 245–251. https://doi. org/10.1021/op900059x 3. Wilcox CS, Pearlman A. Chemistry and Antihypertensive Effects of Tempol and Other Nitroxides. Pharmacol. Rev. 2008, 60 (4), 418–69. https://doi. org/10.1124/pr.108.000240. 4. Yonekuta Y, Oyaizu K, Nishide, H. Structural Implication of Oxoammonium Cations for Reversible Organic One‑Electron Redox Reaction to Nitroxide Radicals. Chem. Lett. 2007, 36 (8), 866–867. 5. Goldstein S, Samuni A. Chemistry of Nitrogen Dioxide and its Biological Implications. Redox Biochem. Chem. 2024, 7, 100020; Soule BP, Hyodo F, Matsumoto K, et al. Free Radic. Biol. Med. 2007, 42, 1632–1650. 6. Lewandowski M., Gwozdzinski K. Nitroxides as Antioxidants and Anticancer Drugs. Int. J. Mol. Sci. 2017, 18 (11), 2490. https://doi.org/10.3390/ijms18112490. 7. Maio N, Lafont, BAP, Sil D, Li Y, Bollinger M, Krebs C. Fe‑S cofactors in the SARS‑CoV‑2 RNA‑dependent RNA Polymerase Are Potential Antiviral Targets. Science 2021, 373 (6551), 236–241. https://doi.org/10.1126/science.abi5224. 8. Bacić G, Nađpal J, Katović V, Šibalić N. Antioxidative Properties of 2, 2, 6, 6‑Tetramethylpiperidine‑N‑oxyl and 2, 6‑di‑tert‑butyl‑4‑methylphenol. Eur. J. Lipid Sci. Technol. 1977, 79 (12), 361–365. 9. Krishna MC, DeGraff W, Hankovszky OH, Russo A. 13C and 15N EPR and ENDOR Studies of Superoxide Dismutase: Tempone and Tempol as Redox Probes of the Enzyme Active Site. Biochemistry 1992, 31 (22), 4996–5002. 10. Soule BP, Hyodo F, Matsumoto KI, et  al. Antioxid. Redox Signal. 2007, 9, 1731–1743. 11. Kagan VE, Jiang JF, Bayir H, Stoyanovsky DA. Free Radic. Biol. Med. 2007, 43, 348–350. 12. Goldstein S, Merenyi G, Russo A, Samuni A. The Role of Oxoammonium Cation in the SOD‑like Activity of Cyclic Nitroxides. J. Am. Chem. Soc. 2003, 125, 789–795. 13. Goldstein S, Samuni A, Hideg K, and Merenyi G. Kinetics of the Reaction Between Nitroxide and Thiyl Radicals: Nitroxides as Antioxidants in the Presence of Thiols. J. Phys. Chem. A 2008, 112, 8600–8605. 14. Goldstein S, Samuni A. Kinetics and Mechanism of Peroxyl Radical Reactions with Nitroxides. J. Phys. Chem. A 2007, 111, 1066–1072. 15. Goldstein S, Fridovich I, Czapski G. Kinetic Properties of Cu, Zn‑superoxide Dismutase as a Function of Metal Content: Order Restored. Free Radic. Biol. Med. 2006, 41, 937–941; Goldstein S, Samuni A, Merenyi G. J. Phys. Chem. A 2008, 112, 8600–8605. 16. Ogura T, Miyoshia A, Koshi M. Rate Coefficients of H‑atom Abstraction from Ethers and Isomerization of Alkoxyalkylperoxy Radicals. Phys. Chem. Chem. Phys. 2007, 9, 5133–5142.

1  •  Introduction to Tempol  17 17. Pavithra D, Ethiraj KR. Synthesis of Xanthenediones and Acridinediones. Polycyclic Aromat. Compd. 2022, 42, 1078. 18. Cai Y, Jalan A, Kubosumi AR, Castle L. Synthesis of 2‑Acylpyrroles. Org. Lett. 2015, 17, 488. 19. Zhang G, Yu Y, Zhao Y, Xie X, Ding C. Synthesis of 2,5‑Disubstituted 1,3,4– Oxadiazole. Synlett. 2017, 28, 1373. 20. Chu X‑Q, Cao W‑B, Xu X‑P, Ji S‑J. Synthesis of Pyrimidines. J. Org. Chem. 2017, 82, 1145. 21. Zhang J‑L, Wu M‑W, Chen F, Han B. Synthesis of Pyrimidines. J. Org. Chem. 2016, 81, 11994. 22. Chen G, Wang Z, Zhang X, Fan X. Synthesis of Pyridines. J. Org. Chem. 2017, 82, 11230. 23. Xu Z‑M, Li H‑X, Young DJ, Zhu D‑L, Li H‑Y, Lang J‑P. Synthesis of Benzothiazoles. Org. Lett. 2019, 21, 237. 24. Lee JW, Lim S, Maienshein DN, Liu P, Ngai MY. Redox‑Neutral TEMPO Catalysis: Direct Radical (Hetero)Aryl C‑H Di‑ and Trifluoromethoxylation. Angew Chem Int Ed Engl. 2020, 59 (48), 21475–21480. doi: 10.1002/ anie.202009490. 25. Zhang G, Li S, Lei J, Zhang G, Xie X, Ding C, Liu R. Biomimetic Aerobic Oxidation of Alcohols. Synlett. 2016, 27, 956. 26. Zhang X‑W, Jiang G‑Q, Lei S‑H, Shan X‑H, Qu J‑P, Kang Y‑B. α,β‑Dehydrogenation. Org. Lett. 2021, 23, 1611. 27. Jiang X, Zhang J, Ma S. Facile and Regioselective Synthesis of N‑Hydroxybenzamides by Palladium‑Catalyzed Aerobic Oxidative Amidation of Acrylamides. J. Am. Chem. Soc. 2016, 138 (27), 8344–8347. 28. Furukawa K, Shibuya M, Yamamoto Y. Oxidation of 1,2‑Diols to α‑Hydroxy Acids. Org. Lett. 2015, 17, 2282. 29. Noh J‑H, Kim J. Aldehyde to Nitrile. J. Org. Chem. 2015, 80, 11624. 30. Zhang G, Xing Y, Xu S, Ring C, Shan S. Synthesis of N‑sulfinyl and N‑sulfonylimines. Synlett. 2018, 29, 1232. 31. Chamorro‑Arenas D, Osorio‑Nieto U, Quintero L, Hernández‑García L, Sartillo‑Piscil F. Synthesis of N‑sulfinyl and N‑sulfonylimines. J. Org. Chem. 2018, 83, 15333. 32. Liu J, Huang J, Jia K, Du T, Zhao C, Zhu R, Liu X. Oxidative Dearomatization. Synthesis. 2020, 52, 763. 33. Zhang Z, Gao Y, Liu Y, Li J, Xie H, Li H, Wang W. Benzylic Oxidation. Org. Lett. 2015, 17, 5492. 34. Xie X, Stahl SS. Catalytic Acceptorless Dehydrogenation (CAD). J. Am. Chem. Soc. 2015, 137, 3767. 35. Wu Y, Yi H, Lei A. Catalytic Acceptorless Dehydrogenation (CAD). ACS Catal. 2018, 8, 1192. 36. Lu W, Shen Z. Dehydrogenative borylation. Org. Lett. 2019, 21, 142.

Synthesis and chemical reactions of Tempol

2

Abhishek Tiwari1*, Varsha Tiwari2*, and Bimal Krishna Banik3*

INTRODUCTION Tempol (TP), or 4‑hydroxy‑TEMPO (2,2,6,6‑tetramethylpiperidine‑1‑oxyl), is a stable nitroxide radical commonly utilized in various fields of chemis‑ try and biology due to its unique redox properties. Its molecular structure is characterized by a piperidine ring substituted with four methyl groups and a nitroxyl (NO) group at the 1‑position, with a hydroxyl group at the 4‑position, conferring significant stability to the radical. Tempol was first synthesized in the mid‑20th century and has since become a critical tool in the study of free radicals and oxidative stress. Its stability and reactivity make it an ideal candidate for applications ranging

Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 2 Department of Pharmacognosy, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 3 Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University, Al Khobar 31952, Kingdom of Saudi Arabia; 1

*

Corresponding Authors: [email protected]; [email protected]; [email protected]

18

DOI: 10.1201/9781003426820-2

2  •  Synthesis and chemical reactions of Tempol  19 from spin labeling in electron paramagnetic resonance (EPR) spectroscopy to its use as a redox‑active agent in biological systems. The synthesis of ­tempol typically begins with the formation of the 2,2,6,6‑tetramethylpiperidine­ skeleton. This can be achieved through various methods, such as the r­ eaction of acetone with ammonia and formaldehyde, followed by cyclization and ­subsequent oxidation. The key step in synthesizing tempol involves the intro‑ duction of the nitroxyl. The synthesis of tempol involves several key steps, including the formation of the piperidine derivative (tempo) and the introduc‑ tion of the hydroxyl group.

Formation of 2,2,6,6‑Tetramethylpiperidine [Tempol (TP)]: Synthesis of tri‑acetoneamine The starting material, 2,2,6,6‑tetramethylpiperidine (TPL), can be synthe‑ sized via cyclization reactions involving appropriate precursors such as ace‑ tone, ammonia, and formaldehyde. TP and derivatives can be synthesized from tri‑acetoneamine) in the presence of NH3/(CH3)2CO and acid catalyst (Figure 1.1, Figure 1) [1].

Mechanism of stabilized radical oxygen of TPL TP, an orange stable radical can be stored at vast temperature for prolonged periods in a normal environment. TP is stabilized through unpaired electron delocalization of nitrogen lone pairs [3–5]. Mechanism of stabilization of TP is depicted in Figure 1.1 (Figure 2).

Synthesis of TPL from tri‑acetoneamine The tri‑acetoneamine can be converted to TP derivatives in the presence of H2O2/Na2WO4/H2O/MeOH continuing the process for 24 hours (Figure 1.1, Scheme1) [2].

Un‑stability mechanism of α‑hydrogens in TPL Some argue that the 4‑CH3 next to NO also creates steric interruption, although TP is active under the correct conditions, the steric impact of these methyl groups is unlikely to be considerable. The methyl groups inhibit self‑decom‑ position by preventing hydrogen distraction, justifying the un‑­stability of NO

20  Chemical and Clinical Applications of Tempol oxides containing alpha hydrogens [6–8]. It is a water‑soluble redox‑cycling nitroxide SOD mimic agent (Figure  1.1, Figure  3) with a low molecular weight that allows it to pass through cell membranes. It has several biologi‑ cal benefits, including radiation protection, metabolic problems, shock, and effects on the heart, kidney, and CNS.

Formation of C–C bond from C–H Indole derivatives exhibit significant biological activities and trimerization of indoles catalyzed by TEMPO and laccases was an efficient method [9]. Recently, the tandem oxidative homocoupling reaction could be achieved by using TEMPO as the catalyst and air as the environmentally benign oxidant in the absence of metal. The trimeric reaction of indoles had broad substrates and high regioselectivity, generating products at the C3 position of indoles (Illustration 1, Scheme 2) [10]. Except for the oxidation of C–H bond to carbonyl compounds, there are also many systems for C–C coupling 10. C(sp3)–H/C(sp3)–H coupling had been achieved using TEMPO as an oxidant and KOtBu as a base under transi‑ tion metal‑free conditions (Illustration 1, Scheme 3). And this approach was suitable for providing 4‑quinolone scaffold through C–C bond formation in excellent yields under mild conditions [11].

Formation of C=O bond from C–H Sartillo‑Piscil reported selective dual C(sp3)–H functionalization at the ­ ‑ and β‑positions of cyclic amines to their corresponding 3‑alkoxyamine α lactams by employing the system including NaClO2/TEMPO/NaClO in either aqueous or organic solvent (Figure  1.1, Scheme 4a). The transition metal free system using TEMPO as the substrate is a simple, mild, and non‑expensive protocol, providing moderate to good yields [12]. Recently, their group continued to achieve dual C(sp3)‑H oxidation of piperazines and morpholines to 2,3‑diketopiperazines and 3 morpholinones catalyzed by TEMPO using NaClO2 and NaOCl as cheap and innocuous reagents (Illustration 1, Scheme 4b). And 2‑alkoxyamino‑3‑morpholinone can be prepared from morpholine derivatives, which would enable further functionalization at the C2 position of the morpholine skeleton by mod‑ ulating the amounts of TEMPO [13]. Besides, construction of vicinal tri‑ carbonyl compounds from 1,3‑­dicarbonyl compounds was realized through DDQ‑mediated oxidative activation of C(sp3)–H bond and subsequent cou‑ pling with TEMPO to form the keyintermediate TEMPO‑substrate adduct (Illustration 1, Scheme 4c) [14].

2  •  Synthesis and chemical reactions of Tempol  21

Oxidation in the presence of TEMPO The reaction uses TEMPO and PhI(OCOCF3)2 (Bis(trifluoroacetoxy) iodobenzene) to selectively oxidize the benzyloxy group at the 4th posi‑ tion of the dihydropyran ring, converting it into a ketone. The combination of these reagents allows for efficient oxidation, leading to the formation of 3‑methoxy‑2,3‑dihydro‑4H‑pyran‑4‑one with a high yield of 86%. TEMPO is capable of abstracting hydrogen atoms from alcohols, forming a nitrosonium ion (TEMPO+) and a radical intermediate on the substrate (Scheme 5) [15–17].

Transformation of ethers Ethers can be transferred into aldehydes, ketones, or nitriles in the presence of TEMPO. A UV (Pyrex filter with a 450‑W medium pressure mercury lamp) light activation/TEMPO oxidation cascade reaction was demon‑ strated to be suitable for the conversion of C–O bond in O‑acetyl aryloxy benzene derivatives to form carbonyl compounds in benzene in the absence of metal (Illustration 1, Scheme 6a) [18]. Hu and coworkers applied DDQ (2,3‑dichloro‑5,6‑dicyano‑1, 4‑benzoquinone)/TEMPO/TBN as a metal‑free catalytic system for direct transformation of PMB (p‑methoxybenzyl) ethers into their corresponding aldehydes or ketones via a new tandem deprotection/ oxidation reaction using oxygen as the oxidant (Illustration 1, Scheme 6b) [19], and alcohols were the important intermediates of the transformation.

Nitration of alkynes To synthesize nitro compounds, nitration of alkenes had been achieved using TEMPO as radical scavenger [18, 20]. In this regard, alkynes have revealed excellent applications. In 2014, Matti’s group presented the stere‑ oselective nitroaminoxylation of alkynes under similar conditions compared with alkenes, which was an efficient approach to functionalized alkynes (Illustration 1, Scheme 7a). Initially, nitro radical from tBuONO oxidized by oxygen in air was added to alkynes. Subsequently, the generated vinyl radi‑ cal was trapped by TEMPO to form the nitration product in high yields [21]. The addition reaction of terminal alkynes was easier than internal alkynes. Hence, it was of great significance to explore reaction of internal alkynes. Li and coworkers proposed the nitrative spirocyclization of alkynes to construct C–N/C–C bonds to produce the difunctionalized spirocyclic product with the same nitro source by employing TEMPO as the initiator (Illustration 1, Scheme 7b) [22]. Besides, metal‑free system including tBuONO and TEMPO for nitrocarbocyclization of 1,6‑enynes was developed by the group of Liang with similar radical mechanism (Illustration 1, Scheme 7c) [23].

22  Chemical and Clinical Applications of Tempol FIGURE 2.1  Figure 1: TPL can be synthesized via cyclization reactions involving acetone, ammonia, and formaldehyde; Figure 2: Mechanism of stabilized radical oxygen of TPL; Scheme 1: Tri‑acetoneamine can be converted into TPL derivatives using NH3 and an acid catalyst; Figure 3: Un‑stability mechanism of α‑hydrogens in TPL; Scheme 2: TEMPO‑mediated reactions are explored for their ability to oxidize C–H bonds, form C–C and C=O bonds, and transform ethers, amines, and sulfur‑containing compounds; Scheme 4: Oxidation in the presence of TEMPO; Scheme 5: Transformation of ethers; Scheme 6: Nitration of alkynes; Scheme 7: Transformation of compounds containing sulfur

2  •  Synthesis and chemical reactions of Tempol  23

Transformation of compounds containing sulfur The methods for synthesizing benzimidazoles and so on mediated by TEMPO have been introduced in other parts of the article. A novel metal‑ and reagent‑free method for the synthesis of benzothiazoles and thiazolopyridines through TEMPO‑catalyzed electrolytic C–H thiolation was exploited (Figure 1.1). In the process, thioamide mediated by TEMPO would be transferred into an inter‑ mediate 8 containing CN bond and S‑O bond, which underwent a cleavage to release two radicals. And thioamidyl radical 9 would go through radical cycliza‑ tion, the loss of electron and proton to form product thiazolopyridines [24]. Yang and coworkers had developed an environmentally friendly approach to furnish 2,2‑dibenzothiazole disulfide from 2‑mercaptobenzothiazole along with TEMPO in the absence of metallic compounds [23, 25]. Furthermore, the construction of sulfur–nitrogen has been demonstrated with TEMPO as the catalyst and O2 as the oxidant in acetonitrile. Thiols could be generated by oxidative homocoupling and heterocoupling reactions [26].

Oxidation reactions using Tempol Various oxidation reactions are illustrated in Figure 2.1 [Scheme 5 (A–K)]. Li and Zhang reported that a TP‑catalyzed reaction using 1‑chloro‑1,2‑ben‑ ziodoxol‑3(1H)—as the intermediate catalyst converts numerous alcohols to their respective carboxylic acids with high to exceptional efficiencies at 350°C in CH3COOC2H5 [27]. Shibuya et al. published their findings that a stable NO radical family of AZADO and 1‑Me‑AZADO outperforms TP as a catalyst, converting alcohols to their respective carboxylic acids in high yields [5(B)] [28]. Zhao and Zhang demonstrated the efficient and non‑toxic conver‑ sion of alcohols to corresponding derivatives using iodobenzene dichloride and pyridine, with large‑scale production methods for iodoarene dichlorides having been developed [5(C)] [29]. In 2001, Luca et  al. demonstrated that primary alcohols could be readily oxidized to their respective aldehydes at room temperature in DCM utilizing tri‑chloroisocyanuric acid (TCCA). The reaction is very chemo‑selective as it takes secondary carbinols a long time to oxidize [5(D)] [30]. Okada et al. reported that NaClO pentahydrate crys‑ tals at extremely low concentrations of NaOH and NaCl could convert pri‑ mary and secondary alcohols to respective carbonyl compounds at constant pH. This novel oxidation technique can also be used for secondary alcohols whose form prevents their utilization [5(E)] [31]. Tamura et  al. reported a straightforward method for producing TP catalysts supported by silica gel. The reaction occurred under moderate conditions, oxidizing alcohols to their respective carbonyl compounds with outstanding yields. The same reagent

24  Chemical and Clinical Applications of Tempol could be employed a minimum of six times [5(F)] [32]. Vatèle et al. reported a fast oxidation method for primary and secondary alcohols using a TP and Yb(OTf)3 mixture along with dodecyl benzene, yielding carbonyl substitutes in exceptional amounts [5(G)] [33]. Ansari and Gree discovered that alde‑ hydes and ketones could be synthesized from primary and secondary alco‑ hols utilizing the TP and copper chloride mixture as catalysts with ILs [bmin] [PF6]. The IL could be utilized many times after washing [5(H)] [34]. Attoui and Vatèle reported the conversion of primary and secondary alcohols in the presence of TP, TBAS, HIO4, and wet alumina [5(I & J)] [35]. Kim and Jung found that the combination of TP and May could be utilized to convert benzylic and allylic alcohols to corresponding carbonyl compounds through aerobic oxidation. Steric hindrance may delay the reaction in allylic systems. This method is better compared to others since reactions occur faster and produce superior outcomes [5(K)] [36]. In 2016, Zhang et al. revealed a new method using FeCl3, L‑valine, and TP for the oxidation of primary and sec‑ ondary benzyl, allylic, and heterocyclic alcohol groups to respective carbonyl groups, yielding excellent results [5(L)] [37]. The oxidation of primary alcohols is detailed in Illustrations 2 [Scheme 6 (A–K)]. Yang et al. revealed that when KBrO3 and hydroxylamine hydrochlo‑ ride react in situ, they generate NOx and Br‑ ions. This allows dioxygen to be activated in the presence of TP, facilitating the oxidation of benzylic alcohols to their respective carbonyl compounds in significant quantities [6(A)] [37]. In 2010, Brioche et al. demonstrated that the Passerini three‑component reac‑ tion can transform alcohols into aldehydes [6(B)] [38]. Zhang et al. devised a combination of Fe(III), L‑valine, and 4–OH–TP to speed up alcohol oxida‑ tion and condensation with sulfinamide/sulfonamide, synthesizing N‑sulfinyl/ N‑sulfonyl imines [6(C)] [39]. Zhang et  al. examined the iron‑catalyzed aerobic oxidation of primary and secondary amines, including benzylamines and anilines [6(D)] [40]. Yin et al. showed that nitriles can be directly pro‑ duced from alcohols and NH4OH in a mild, aerobic, and catalytic fashion. This procedure also enabled the production of several biaryl heterocycles from commercially available alcohols in a single vessel [6(E)] [41]. Bolm et al. demonstrated a metal‑free oxidation synthesis of carbonyl compounds by TP, where ketone synthesis was very effective due to moderate conditions. Oxone can function even with silyl protecting groups that it would ordinarily break apart [6(F)] [42]. Jiang and Ragauskas noted that the conversion of alcohols to carboxylic acids in the presence of Fe(NO3)3.9H2O/TP/MCl results in high yield [6(G)] [43]. In 2005, Jiang and Ragauskas revealed the conversion of alcohols to their respective carboxylic acids in the presence of air [6(H)] [44]. Jiang and Ragauskas reported a four‑step process involving acetamido‑TP, DABCO, TMDP, and Cu(ClO4)2 in DMSO, which allows aerobic oxidation of alcohols to their corresponding carboxylic acids with excellent results, and the process is recyclable [6(I)] [45]. Hoover et  al. described a high‑yielding

2  •  Synthesis and chemical reactions of Tempol  25

FIGURE  2.2  Examples of specific reactions include the transformation of alcohols to carboxylic acids, conversion of primary amines to nitriles, and oxidative synthesis of quinazolines.

26  Chemical and Clinical Applications of Tempol ­oxidative method for converting alcohols to carboxylic acid derivatives [6(J)] [46]. Gheorghe et al. [47] reported the use of TP with multiple perfluoroalkyl and triazole for the conversion of alcohols, with the solvent recovered through a filtration technique [6(K)] [47] (Figure 2.2). Recently, Ji focused on selenium functionalization of indoles and synthe‑ sized a series of 3‑selenylindole derivatives catalyzed by TEMPO with O2 as the green oxidant and selenium powder as the selenium source. Electron spin resonance (ESR) studies revealed that this approach involved the formation of nitrogen‑centered radicals and selenium radicals via oxidation of in situ generated selenoates (Illustration 3, Scheme 8) [48]. Tertiary anilines are more stable, and researchers often employ transition metal‑catalyzed activation of inert chemical bonds (C–N bond) [49]. Xiaodong Jia reported that a catalytic metal‑free system including TBN (tert‑butyl nitrite)/TEMPO was developed for highly selective C(sp3)–N cleav‑ age of tertiary anilines, exhibiting high efficiency and good functional group tolerance under mild reaction conditions (Illustration 3, Scheme 9) [50]. Amines, a class of widely used substances, are acutely sensitive to oxida‑ tion, producing different products depending on the oxidant. The conversion of a primary amine to nitrile is particularly challenging. Tetrahydropyridazines, an important class of six‑membered heterocycles, are found in many natu‑ ral products and pharmaceutically active compounds. Yang and coworkers reported a one‑pot tandem reaction including oxidative dehydrogenation of ketohydrazones and subsequent aza‑Diels‑Alder reaction for synthesizing tetrahydropyridazines in the presence of TEMPO, which acts as a radical initiator (Illustration 3, Scheme 10) [51]. Likhtenshtein and coauthors reported the oxidation of primary amines in good yields mediated by a stoichiometric quantity of 4‑acetamido‑2,2,6,6‑tet‑ ramethylpiperidine‑1‑oxoammonium tetrafluoroborate as the oxidant in CH2Cl2‑pyridine solvent at room temperature or gentle reflux (Illustration 3, Scheme 11a) [52, 53]. The preparation of carbonyl compounds from nitriles is notable. The transformation of primary and secondary amines to carbonyl compounds can be achieved using PhI(OAc)2 as the oxidant and TEMPO as the catalyst under mild conditions (Illustration 3, Scheme 11b) [54]. Moreover, 2‑substituted benzoxazoles, benzothiazoles, and benzimid‑ azoles can be prepared by oxidative dehydrogenation catalyzed by 4‑methoxy TEMPO, undergoing a one‑pot reaction between aldehydes and 2‑aminophe‑ nol, 2‑aminothiophenol, or o‑phenylenediamine, respectively (Illustration 3, Scheme 11c) [53]. Similarly, primary amines can react with aldehydes to generate pyrrolo[1,2‑a]quinoxalines initiated by TEMPO oxoammonium salts, driving a Pictet–Spengler reaction of imine to undergo cyclization‑dehydrogenation for the formation of quinoxalines (Illustration 3, Scheme 11d) [53].

2  •  Synthesis and chemical reactions of Tempol  27 A novel and efficient aerobic protocol for the oxidative synthesis of 2‑aryl quinazolines via benzyl C–H bond amination by a one‑pot reaction of arylmethanamines with 2‑aminobenzoketones and 2‑aminobenzaldehydes catalyzed by 4‑hydroxy‑TEMPO without the need for metals or other addi‑ tives (Illustration 3, Scheme 11e) [53]. N3‑radicals resulting from N3‑iodine(III) reagent with the help of TEMPONa can react with alkenes to provide the corresponding C‑radicals, yielding products in good to excellent yields under mild conditions (Illustration 3, Scheme 12a) [53, 54]. Aryl radicals generated from aryl diazonium or hypervalent iodine(III) compounds can add to alkenes, with subsequent TEMPO trapping providing the corresponding oxyarylation products in good to excellent yields (Scheme 12b) [53, 55]. Additionally, NFSI can be reduced to an N‑centered radical by TEMPONa, which reacts with alkenes to give aminooxygenation products in moderate to good yields (Scheme 12c) [53, 56]. Moreover, Studer’s group found that the hypervalent iodine‑CF3 reagent (Togni reagent) can be trans‑ formed into the CF3 radical, which can be trapped by alkenes (Illustration 3, Scheme 12d) [53] (Figure 2.3).

CONCLUSION 2,2,6,6‑Tetramethylpiperidine (TPL), commonly known as Tempol (TP), is a versatile compound widely utilized in organic synthesis, catalysis, and chemical transformations. This conclusion highlights the diverse applications and significance of TPL in various chemical processes. TPL can be synthe‑ sized from precursors like acetone, ammonia, and formaldehyde through cyclization reactions. Its stability, attributed to the delocalization of nitrogen lone pairs, enables prolonged storage under various conditions, making it a valuable reagent in organic chemistry. TPL acts as an efficient catalyst in numerous oxidation reactions, facilitating the conversion of alcohols to car‑ boxylic acids, aldehydes, or ketones with high efficiency and selectivity. It participates in C–C and C=O bond formations from C–H bonds, enabling the synthesis of diverse organic compounds. TPL plays a crucial role in ste‑ reoselective nitroaminoxylation reactions of alkynes and the construction of C–N/C–C bonds. Additionally, it enables the synthesis of sulfur‑containing compounds like benzothiazoles and thiazolopyridines through electrolytic C–H thiolation. In amine transformations, TPL facilitates oxidative dehydro‑ genation reactions and subsequent transformations to form heterocycles like tetrahydropyridazines. It also enables selenium functionalization of indoles and selective C(sp3)–N cleavage of tertiary anilines. TPL’s versatility in

28  Chemical and Clinical Applications of Tempol

FIGURE 2.3  Various figures and schemes are referenced to illustrate the detailed reaction mechanisms and outcomes.

2  •  Synthesis and chemical reactions of Tempol  29 catalyzing a wide range of reactions, its stability, and its ability to facilitate selective transformations make it a valuable tool in organic synthesis. It offers mild reaction conditions, high yields, and functional group tolerance, mak‑ ing it attractive for synthetic chemists. In conclusion, TPL, with its unique properties and catalytic capabilities, holds significant promise in advancing organic synthesis and chemical transformations. Further research into its applications and optimization of reaction conditions could lead to enhanced synthetic methodologies and the discovery of novel compounds with diverse functionalities and applications.

REFERENCES

1. Vaz SM, Augusto O. The Mechanism by Which TEMPOL Inhibits Peroxidase‑ mediated Protein Nitration. Free Radic. Biol. Med. 2006, 41S, S142. 2. Process for preparing 2,2,6,6‑tetramethyl‑4‑piperidone (1978) US4536581A Patent, United States. 3. Shibuya MI, Tomizawa S, Iwabuchi Y. 2‑Azaadamantane N‑Oxyl (AZADO) and 1‑Me‑AZADO:  Highly Efficient Organocatalysts for Oxidation of Alcohols. JACS 2006, 128, 8412–8413. https://doi.org/10.1021/ja0620336. 4. Laight DW, Andrews TJ, Haj‑Yehia AI, Carrier MJ, Anggard EE. Microassay of superoxide anion scavenging activity in vitro. Environ. Toxicol. Pharmacol. 1997, 3 (1), 65–68. https://doi.org/10.1016/s1382‑6689(96)00143‑3. 5. Wilcox CS. Effects of TEMPOL and Redox‑cycling Nitroxides in Models of Oxidative Stress. Pharmacol. Ther. 2010, 126 (2), 119–145. https://doi. org/10.1016/j.pharmthera.2010.01.003. 6. Khattab MM. Tempol, a Membrane‑Permeable Radical Scavenger, Attenuates Peroxynitrite‑ and Superoxide Anion Enhanced Carrageenan‑Induced Paw Edema and Hyperalgesia: A Key Role for Superoxide Anion. Eur. J. Pharmacol. 2006, 548 (1–3), 167–173. https://doi.org/10.1016/j.ejphar.2006.08.007. 7. Tal M. A Novel Antioxidant Alleviates Heat Hyperalgesia in Rats with an Experimental Painful Peripheral Neuropathy. NeuroReport 1996, 7 (8), 1382– 1384. https://doi.org/10.1097/00001756‑199605310‑00010. 8. Joseph PA. New Applications of TP in Organic Synthesis. University of York, Chemistry PhD thesis, 2015. 9. Zu X, Wang YF, Ren W, Zhang FL, Chiba S. TP‑Mediated Aliphatic C‑H Oxidation with Oximes and Hydrazones. Org. Lett. 2013, 15, 3214–3214. https://doi.org/10.1021/ol4014969 10. Han B, Yang XL, Wang C, Bai YW, Pan TC, Chen X, Yu W. CuCl/ DABCO/4‑HO‑TP‑Catalysed Aerobic Oxidative Synthesis of 2‑Substituted Quinazolines and 4H‑3,1‑Benzoxazines. J. Org. Chem. 2012, 77, 1136–1142. https://doi.org/10.1021/jo2020399 11. Chen Z, Chen J, Liu M, Ding, J, Gao W, Huang X, Wu H. Unexpected CopperCatalysed Cascade Synthesis of Quinazoline Derivatives. J. Org. Chem. 2013, 78, 11342–11348. https://doi.org/10.1021/jo401908g

30  Chemical and Clinical Applications of Tempol 12. Zhang JL, Wu MW, Chen F, Han B. Cu‑Catalysed [3+3] Annulation for the Synthesis of Pyrimidines via β‑C(sp3)–H Functionalization of Saturated Ketones. J. Org. Chem. 2016, 81 (23), 11994–12000. https://doi.org/10.1021/ acs.joc.6b02181. 13. Ding X, Qiu Y. Long Metal‑Free TEMPO‑Promoted C(sp3)–H Amination to afford multi‑substituted Benzimidazoles. J. Org. Chem. 2014, 79 (1), 4727–4734. 14. Xu ZM, Li HX, Young DJ, Zhu DL, Ha YL, Lang JP. Exogenous Photosensitizer‑Metal‑, and Base‑Free Visible‑Light‑Promoted C–H Thiolation via Reverse Hydrogen Atom Transfer. Org. Lett. 2019, 21 (1), 237–241. https:// doi.org/10.1021/acs.orglett.8b03679. 15. Hejun A, Shaoyu M, Qingqing X, Yao Z, Qiuling S. Gold‑Catalyzed Radical‑Involved Intramolecular Cyclization of Internal N ‑Propargylamides for the Construction of 5‑Oxazole Ketones. J. Org. Chem. 2018, 84, https://doi. org/10.1021/acs.joc.8b02334. 16. Lin JP, Zhang FH, Long YQ. Solvent/Oxidant‑Switchable Synthesis of Multisubstituted Quinazolines and Benzimidazoles via Metal‑Free Selective Oxidative Annulation of Arylamidines. Org. Lett. 2014, 16: 2822–2825. https:// doi.org/10.1021/ol500864r 17. Chu XQ, Cao WB, Xu XP, Ji SJ. Iron Catalysis for Modular Pyrimidine Synthesis through β‑Ammoniation/Cyclization of Saturated Carbonyl Compounds with Amidines. J. Org. Chem. 2017, 82 (2), 1145–1154. https://doi.org/10.1021/acs. joc.6b02767. 18. Xie X, Stahl SS. Efficient and Selective Cu/Nitroxyl‑Catalysed Methods for Aerobic Oxidative Lactonization of Diols. JACS 2015, 137 (11), 3767–3770. 19. Pradhan PP, Bobbitt JM, Bailey WF. Oxidative Cleavage of Benzylic and Related Ethers, Using an Oxoammonium Salt. J. Org. Chem. 2009, 74, ­9501–9504. https://doi.org/10.1021/jo902144b. 20. Cui Z, Du DM. Enantioselective Synthesis of β‑Hydrazino Alcohols Using Alcohols and NBoc‑Hydrazine as Substrates. Org. Lett. 2016, 18, 5616–5619. https://doi.org/10.1021/acs.orglett.6b02841 21. Naveen T, Maity S, Sharma U, Maiti D. A Predictably Selective Nitration of Olefin with Fe(NO3)3 and TP. J. Org. Chem. 2013, 78, 5949–5954. https://doi. org/10.1021/jo400598p. 22. Chen FE, Kuang YY, Dai HF, Lu L, Huo M. A Selective and Mild Oxidation of Primary Amines to Nitriles with Trichloro‑isocyanuric Acid. Synthesis 2003, 2629–2631. https://doi.org/10.1055/s‑2003‑42431. 23. Lu W, Shen Z. Direct Synthesis of Alkenyl‑boronates from Alkenes and Pinacol Diboron via Copper Catalysis. Org. Lett. 2019, 21, 142–146. https://doi. org/10.1021/acs.orglett.8b03599 24. Khan IA, Saxena AK. Metal‑Free, Mild, Nonepimerizing, Chemo‑ and Enantio‑ or Diastereoselective N‑Alkylation of Amines by Alcohols via Oxidation/ Imine–Iminium Formation/Reductive Amination: A Pragmatic Synthesis of Octahydropyrazinopyridoindoles and Higher Ring Analogues. J. Org. Chem. 2013, 78, 11656–11669. https://doi.org/10.1021/jo4012249 25. Shibuya M, Tomizawa M, Suzuki I, Iwabuchi Y. 2‑Azaadamantane N‑Oxyl (AZADO) and 1‑Me‑AZADO:  Highly Efficient Organocatalysts for Oxidation of Alcohols. JACS 2006, 128, 8412–8413. https://doi.org/10.1021/ja0620336

2  •  Synthesis and chemical reactions of Tempol  31 26. 1Zhao XF, Zhang C. An Environmentally Benign TP‑Catalysed Efficient Alcohol Oxidation System with a Recyclable Hypervalent Iodine (III) Reagent and Its Facile Preparation. Synthesis 2007, 551–557. https://doi. org/10.1055/s‑2007‑965889. 27. Li XQ, Zhang C. An Environmentally Benign TP‑catalysed efficient alco‑ hol oxidation system with a recyclable hypervalent iodine (III) reagent and its facile preparation. Synthesis 2009, 1163–1169. https://doi.org/10.1055/s‑ 0028‑1087850. 28. Shibuya M, Tomizawa M, Suzuki I. Iwabuchi Y.2‑Azaadamantane N‑Oxyl (AZADO) and 1‑Me‑AZADO:  Highly Efficient Organocatalysts for Oxidation of Alcohols. JACS 2006, 128, 8412–8413. https://doi.org/10.1021/ja0620336 29. Zhao XF, Zhang C. An Environmentally Benign TP‑Catalysed Efficient Alcohol Oxidation System with a Recyclable Hypervalent Iodine (III) Reagent and Its Facile Preparation. Synthesis 2007, 551–557. https://doi.org/10.1055/ s‑0028‑1087850 30. Luca LD, Giacomelli G, Porcheddu A. A Very Mild and Chemoselective Oxidation of Alcohols to Carbonyl Compounds. Org. Lett. 2001, 3, 3041–3043. https://doi.org/10.1021/ol016501m 31. Okada T, Asawa T, Sugiyama Y, Kirihara M, Iwai T, Kimura Y. Sodium hypo‑ chlorite pentahydrate (NaOCl·5H2O) Crystals as an Extra­ordinary Oxidant for Primary and Secondary Alcohols. Synlett. 2014, 25, 596–598. https://doi. org/10.1055/s‑0033‑1340483. 32. Tamura N, Aoyama T, Takido T, Kodomari M. Novel [4‑Hydroxy‑TP + NaCl]/ SiO2 as a Reusable Catalyst for Aerobic Oxidation of Alcohols to Carbonyls. Synlett. 2012, 23, 1397–1407. https://doi.org/10.1055/s‑0031‑1290980 33. Vatèle JM. Yb (OTf)3‑Catalysed Oxidation of Alcohols with Iodosylbenzene Mediated by TP. Synlett. 2006, 2055–2058. https://doi.org/10.1055/s‑2006‑948181. 34. Ansari IA, Gree R. TP‑Catalysed Aerobic Oxidation of Alcohols to Aldehydes and Ketones in Ionic Liquid [bmin][PF6]. Org. Lett. 2001, 1507–1509. https:// doi.org/10.1021/ol025721c. 35. Attoui M, Vatèle JM. TP/NBu4Br‑Catalysed Selective Alcohol Oxidation with Periodic Acid. Synlett. 2014, 25, 2923–2927. https://doi.org/10.1055/s‑0034‑1378913 36. Kim SS, Jung HC. An Efficient Aerobic Oxidation of Alcohols to Aldehydes and Ketones with TP/Ceric ammonium nitrate as catalysts. Synthesis 2003, 2135–2137. https://doi.org/10.1055/s‑2003‑41065 37. Yang G, Wang W, Zhu W, An C, Gao X, Song M. In situ Formation of NOx and Br Anion for Aerobic Oxidation of Benzylic Alcohols without Transition Metal. Synlett. 2010, 437–440. https://doi.org/10.1055/s‑0029‑1219202 38. Brioche J, Masson G, Zhu JP. Three‑Component Reaction of Alcohols under Catalytic Aerobic Oxidative Conditions. Org. Lett. 2010, 12, 1432–1435. https:// doi.org/10.1021/ol100012y 39. Zhang G, Xing Y, Xu S, Ring C, Shan S. Fe(III)/l‑Valine‑Catalysed One‑Pot Synthesis of N‑Sulfinyl‑ and N‑Sulfonylimines via Oxidative Cascade Reaction of Alcohols with Sulfinamides or Sulfonamides. Synlett. 2018, 29, 1232. https:// doi.org/10.1055/s‑0037‑1609320. 40. Zhang E, Tian H, Xu S, Yu X, Xu Q. Iron‑Catalysed Direct Synthesis of Imines from Amines or Alcohols and Amines via Aerobic Oxidative Reactions under Air. Org. Lett. 2013, 15, 2704–2707. https://doi.org/10.1021/ol4010118.

32  Chemical and Clinical Applications of Tempol 41. Yin W, Wang C, Huang H. Highly Practical Synthesis of Nitriles and Heterocycles from Alcohols under Mild Conditions by Aerobic Double Dehydrogenative Catalysis. Org. Lett. 2013, 15, 1850–1853. https://doi.org/10.1021/ol400459y. 42. Bolm C, Magnus AS, Hildebrand JP. Catalytic Synthesis of Aldehydes and Ketones under Mild Conditions Using TP/Oxone. Org. Lett. 2000; 2: 1173– 1175. https://doi.org/10.1021/ol005792g. 43. Jiang N, Ragauskas AJ. Copper (II)‑Catalysed Aerobic Oxidation of Primary Alcohols to Aldehydes in Ionic Liquid [bmpy] PF6. Org. Lett. 2005, 7, 3689– 3692. https://doi.org/10.1021/ol051293+. 44. Jiang N, Ragauskas AJ. Cu (II)‑Catalysed Selective Aerobic Oxidation of Alcohols under Mild Conditions. J. Org. Chem. 2006, 71, 7087–7090. https:// doi.org/10.1021/jo060837y. 45. Hoover JM, Ryland BL, Stahl SS. Copper/TP‑Catalysed Aerobic Alcohol Oxidation: Mechanistic Assessment of Different Catalyst Systems. ACS Catal. 2013, 3 (11), 2599–2605. https://doi.org/10.1021/cs400689a 46. Benaglia M, Puglisi A, Cozzi F. Polymer‑supported Organic Catalysts. Chem. Rev. 2003, 103, 3401–3430. https://doi.org/10.1021/cr010440o47. Gheorghe A, Chinnusamy T, Cuevas‑Yañez E, Hilgers P, Reiser O. Combination of Perfluoroalkyl and Triazole Moieties: A New Recovery Strategy for TEMPO. Org. Lett., 2008, 10, 4171–4174. 48. Liu H, Fang Y, Wang SY, Ji SJ. TEMPO‑Catalyzed Aerobic Oxidative Selenium Insertion Reaction: Synthesis of 3‑Selenylindole Derivatives by Multicomponent Reaction of Isocyanides, Selenium Powder, Amines, and Indoles under Transition‑Metal‑Free Conditions. Org. Lett. 2018, 20, 4, 930– 933. https://doi.org/10.1021/acs.orglett.7b03783 49. Wang Q, Lixin YS Li, Huang H. Transition‑Metal Catalysed C–N Bond Activation. Chem. Soc. Rev., 2016, 45, 1257–1272. https://doi.org/10.1039/ C5CS00534E 50. Jia X, Li P, Shao Y, Yuan Y, Ji H, Hou W, Liu X, Zhang X. Highly Selective sp3 C–N Bond Activation of Tertiary Anilines Modulated by Steric and Thermodynamic Factors. Green Chem. 2017, 19, 5568–5574. https://doi. org/10.1039/C7GC02775C 51. Peng XX, Chen F, Han B. TEMPO‑Mediated Aza‑Diels‑Alder Reaction: Synthesis of Tetrahydropyridazines Using Ketohydrazones and Olefins. Org. Lett. 2016, 18 (9), 2070–2073. https://doi.org/10.1021/acs.orglett.6b00702 52. Likhtenshtein GI. Nitroxide Chemical Reactions. In Nitroxides. Springer Series in Materials Science, vol. 292. Springer: Cham. https://doi. org/10.1007/978‑3‑030‑34822‑9_3 53. Bansodeab AH, Suryavanshi G. Metal‑free Hypervalent Iodine/TEMPO Mediated Oxidation of Amines and Mechanistic Insight into the Reaction Pathways. RSC Adv. 2018, 8, 32055–32062. https://doi.org/10.1039/C8RA07451H 54. Xi Wang, Armido Studer. Iodine(III) Reagents in Radical Chemistry. Acc. Chem. Res. 2017, 50, 7, 1712–1724. https://doi.org/10.1021/acs.accounts.7b00148 55. Macias CA, Chiao JW, Xiao J, Arora DS, Tyurina YY, Delude RL, Wipf P, Kagan VE, Fink MP. Treatment with a Novel Hemigramicidin‑TEMPO Conjugate Prolongs Survival in a Rat Model of Lethal Hemorrhagic Shock. Ann. Surg. 2007, 245, 305–314. https://doi.org/10.1097/01.sla.0000236626.57752.8e 56. Vaz SM, Augusto O. The Mechanism by Which TEMPOL Inhibits Peroxidase‑ Mediated Protein Nitration. Free Radic. Biol. Med. 2006, 41S, S142.

Tempol in the synthesis of terpenoids

3

Abhishek Tiwari1*, Varsha Tiwari2*, and Bimal Krishna Banik3*

INTRODUCTION The synthesis of terpenoids, a diverse class of natural compounds renowned for their wide array of biological activities and industrial applications, has long been a focal point of organic chemistry research. These molecules, characterized by their complex carbon skeletons derived from isoprene units, exhibit remarkable structural diversity and functional versatility, making them valuable targets for synthetic endeavors [1]. In recent years, the application of transition metal‑catalyzed processes has revolutionized the field of terpenoid synthesis, offering efficient and selec‑ tive routes to complex molecular architectures. Among the myriad of cata‑ lysts explored, 2,2,6,6‑tetramethylpiperidine‑1‑oxyl (TEMPO) has emerged Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 2 Department of Pharmacognosy, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 3 Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University, Al Khobar 31952, Kingdom of Saudi Arabia; 1

*

Corresponding Authors: [email protected]; [email protected]; [email protected]

DOI: 10.1201/9781003426820-3

33

34  Chemical and Clinical Applications of Tempol as a powerful tool for the synthesis of terpenoids, owing to its unique ability to facilitate selective oxidation reactions [2].

FEW SYNTHESIS OF TERPENOIDS USING TEMPO Homoallyl alcohol (1) was achieved by removing the protecting group, following TEMPO‑interceded oxidation, the addition of 3‑lithio furan to the synthesized aldehyde, and DMP oxidation yielded the furanyl ketone. The Corey–Bakshi reduction in furanyl ketone (2) following (S)‑2‑methyl CBS‑oxa‑zaborolidine and (R) 2‑methyl CBS‑oxa‑zaborolidine synthesized the (S)‑alcohol (–)‑(3) in a 73% yield. Deprotection of both enantiomeric alcohols (–)‑3 with tetra‑butyl ammonium fluoride and the subsequent region‑selective oxidation of the 3‑alkyl furan with [O] in the manifestation of Hünig’s base generated (–)‑Aplysinoplide B (4) in a 48% yield (1) (Figure 3.1, Scheme 1) [3].

TOTAL SYNTHESIS OF (–)‑APLYSINOPLIDE B Total synthesis of hyperjapone‑A started with Friedel–Crafts acylation of compound phloroglucinol with isobutyryl chloride to provide acyl phloro‑ glucinol, which was dearomatized using trimethylation to yield compound norflavesone (2 steps). Oxidation of norflavesone with Ag2O and TEMPO gave hyperjapone‑A in a 32% yield, through a hetero Diels–Alder among the α, β‑unsaturated ketone, generated instantly, and humulene (Figure 3.1, Scheme 2). Treatment of hyperjapone‑A (±) with m‑CPBA gave epoxide as a major diastereomer in a 76% yield over diastereoselective oxidation of the Δ 8, 9 alkenes. Acid‑mediated rearrangement of epoxide following p‑TsOH in CH2Cl2 afforded hyperjaponol‑C in a 43% yield. Transformation of epoxide into (±)‑hyperjaponol‑A was obtained in a 59% yield by treating LiBr and (NC)2C=C(CN)2 in acetone (Figure 3.1, Scheme 3) [4–7].

SYNTHESIS OF HYDROXYSTEROIDS Shen and co‑workers [8] performed great work on the oxidation of sensitive hydroxysteroids to their respective ketosteroids by utilizing a mixture of DDQ

3  •  Tempol in the synthesis of terpenoids  35

FIGURE  3.1  Scheme 1: Total synthesis of (–)‑aplysinoplide B; Scheme 2: Total synthesis of hyperjapone‑A; Scheme 3: Diastereoselective oxidation and transformation of hyperjapone‑A; Scheme 4: Oxidation of hydroxysteroids to ketosteroids

36  Chemical and Clinical Applications of Tempol and a catalytic amount of tetramethyl piperidinyl‑1‑oxyl (TEMPO) reagent as an effective oxidative system (Figure 3.1, Scheme 4). These ­oxidative con‑ ditions proved a high yielding. This procedure was most effective for the preparation of 4,6‑diene‑3‑one from the corresponding hydroxysteroids [9].

SYNTHESIS OF PARTHENOLIDE Scheme 5 details the procedures for synthesizing parthenolide. The initial step involves creating the unsaturated sulfonyl amide via a two‑step process: a Horner– Wadsworth–Emmons reaction with a ketone and di‑­ethylphosphonoacetic acid. Using titanium tetrachloride (TiCl4) and di‑isopropylethylamine (i‑Pr2NEt) in dichloromethane (CH2Cl2), an aldol reaction with this compound and an aldehyde yields the primary product with the desired 6,7‑stereochemistry. Selective cleav‑ age of the tert‑butyldimethylsilyl ether (TBS) protecting group of this compound using HCl in ethanol at 0°C, followed by treatment with 2‑­methoxypropene, produces the acetonide. A reduction reaction then yields the thioether prod‑ uct, which is subsequently treated with diphenyl disulfide/tri‑n‑butylphosphine (Figure 3.2, Scheme 5). Oxidation in tert‑butanol (t‑BuOH) and pyridine using hydrogen peroxide/ammonium heptamolybdate (H2O2/(NH4)6Mo7O24) follows. Tetrabutylammonium fluoride removes the tert‑­butyldiphenylsilyl ether (TBDPS) group, yielding an alcohol [10–12]. At 0°C, alcohol conversion to its corre‑ sponding brominated molecule is achieved using tetrabromomethane (CBr4), triphenylphosphine (PPh3), and 2,6‑lutidine as bases. The desired cyclized prod‑ uct is obtained by treating this chemical with four equivalents of potassium bis(trimethylsilyl)amide (KHMDS). The sulfone moiety on the cyclized product is then eliminated by adding magnesium/methanol (Mg/MeOH), resulting in the product [13]. Pyridinium p‑toluenesulfonate in methanol successfully removes the acetonide group, forming the required ten‑membered carbocyclic germacrene ring intermediate. Parthenolide is obtained through Sharpless epoxidation of the diol, followed by oxidation with 2,2,6,6‑tetramethyl‑1‑piperidinyloxy (TEMPO) and (diacetoxyiodo)benzene (PhI(OAc)2) [14].

PARTHENOLIDE SEMI‑SYNTHESIS Large and complex compounds obtained from natural sources are frequently used as starting materials in the semi‑synthesis strategy. This approach is particularly useful when the precursor molecule includes a structurally

3  •  Tempol in the synthesis of terpenoids  37 complex component that is either too expensive or too difficult to synthesize via complete synthesis. The simplest technique for synthesizing a complex natural product is to begin with molecules that already possess the necessary germacranolide skeleton and then synthesize the target molecule through a sequence of chemical modifications. Parthenolide is synthesized using a protection‑free technique starting from the natural product costunolide (Figure  3.2, Scheme 6). Costunolide, which is easily extracted from the roots of Saussurea lappa, has been identi‑ fied as an excellent substrate for the synthesis of parthenolide. To obtain the

FIGURE  3.2  Scheme 5: Synthesis of parthenolide; Scheme 6: Parthenolide semi‑synthesis

38  Chemical and Clinical Applications of Tempol crucial germacrane intermediate, costunolide is treated with diisobutylalumi‑ num hydride (DIBAL) in toluene at room temperature. The protected primary alcohol is then obtained in good yield by treatment with tert‑butyldimethylsilyl chloride (TBSCl). This intermediate is further processed via selective epoxi‑ dation of the C4 –C5 bond in CH2Cl2 at room temperature using titanium isop‑ ropoxide (Ti(Oi‑Pr)4), (–)‑diisopropyl D‑tartrate (D‑(–)‑DIPT), and tert‑butyl hydroperoxide (TBHP). Parthenolide is finally obtained by deprotecting this compound and then oxidizing it with TEMPO and PhI(OAc)2 [15–17].

CHIRAL SYNTHESIS OF SELECTED TERPENOIDS Synthesis of (+)‑apiosporamide This is an example of the intramolecular Diels‑Alder (IMDA) reaction and its transannular modification. To synthesize the antibiotic alkaloid (+)‑apiosporamide, two advanced intermediates from the chiral carbon pool are combined: an amino acid derived from quinic acid and trans‑decalin derived from citronellol (Figure 3.3, Scheme 7) [15–17]. In the first step, quinic acid is used to make cyclohexenone, which is then deoxygenated to produce an intermediate. Deprotonated β‑lactam is added, resulting in an adduct, which is then isolated and stereoselectively trans‑ formed into an epoxide. Allyl alcohol is used to open the lactam, producing the necessary amino acid derivative [15–17]. In a parallel process, citronellol is transformed into an alkyne, which is then employed to produce the Diels‑Alder precursor through a Negishi‑type chain elongation. High endo‑selectivity IMDA is used to produce the trans‑decalin ketone, which is then carboxylated to produce an acid. The allyl‑protecting group is removed after coupling with the intermediate, fol‑ lowed by carbonyl activation. A keto lactam is produced via Dieckmann ring closure, then deprotected and aromatized to produce the final product [15–17].

Synthesis of hirsutellone B from (+)‑citronellal (+)‑Citronellal has been utilized in IMDA reactions, producing the fun‑ gal metabolite hirsutellone B (Scheme 8). (R)‑Citronellal is transformed

3  •  Tempol in the synthesis of terpenoids  39 into epoxy vinyl iodide, which is then combined with stannane in a Stille cross‑coupling to produce polyene. In the presence of Et2AlCl, the stannane engages in a Sakurai‑type cyclization with the epoxide to produce the cyclo‑ hexane derivative, which is then subjected to a tandem IMDA reaction, annu‑ lating two additional rings. Thus, the stereoselective synthesis of the tricyclic intermediate is accomplished. The next subgoal involves the transformation into sulfone. After functionalizing the aryl methyl group, the aldehyde pro‑ duced via Mukaiyama etherification and reduction of the ester is employed to extend the sidechain and produce the ketone. The introduction of a pri‑ mary iodide and a thioacetate produces the intermediate, which under basic conditions cyclizes to the thioether. Sulfone is produced by oxidation. The (Z)‑cycloolefin is produced by the Ramberg‑Bäcklund ring contraction and converted to the β‑keto ester by carboxylation. Alcohol is produced stereose‑ lectively using Sharpless asymmetric dihydroxylation (AD) of the olefin and regioselective Barton‑McCombie deoxygenation of the diol. After the alcohol is converted to a ketone, it is heated with NH3 to produce the final product through an amidation‑epimerization cyclization cascade of C‑17 (Figure 3.3, Scheme 8) [15–17].

CONCLUSION The synthesis of terpenoids utilizing TEMPO as a catalyst represents a dynamic and rapidly evolving field within organic chemistry. From its incep‑ tion as a tool for selective oxidation reactions to its diverse applications in terpenoid synthesis, TEMPO has played a pivotal role in advancing our understanding and capabilities in this area. The total synthesis of complex terpenoids, such as (–)‑aplysinoplide B and (±)‑hyperjapone‑A, along with their racemic analogs, stands as a testament to the power and versatility of TEMPO‑mediated transformations. These endeavors have not only enabled the efficient construction of intricate molecular architectures but have also provided valuable insights into the synthetic strategies and reactivity patterns involved. While successful transformations have showcased the efficacy of TEMPO in achieving desired structural modifications, challenges such as unsuccessful C10 oxidation attempts have underscored the need for further exploration and optimization of reaction conditions. These setbacks serve as valuable learning experiences, driving the refinement of synthetic method‑ ologies and the development of novel approaches. The synthesis of biologically significant compounds like parthenolide and (+)‑apiosporamide from citronellol highlights the practical applications

40  Chemical and Clinical Applications of Tempol

FIGURE 3.3  Scheme 7: Synthesis of (+)‑apiosporamide; Scheme 8: Synthesis of hirsutellone B from (+)‑citronellal

3  •  Tempol in the synthesis of terpenoids  41 of TEMPO‑catalyzed reactions in drug discovery and natural product chem‑ istry. Whether through total synthesis, semi‑synthesis, or derivatization strat‑ egies, TEMPO continues to facilitate efficient access to diverse terpenoid scaffolds with potential therapeutic relevance.

REFERENCES

1. Masyita A, Mustika Sari R, Dwi Astuti A, Yasir B, Rahma Rumata N, Emran TB. Terpenes and Terpenoids as Main Bioactive Compounds of Essential Oils, Their Roles in Human Health and Potential Application as Natural Food Preservatives. Food Chem. X. 2022 Jan 19, 13, 100217. https://doi.org/10.1016/j. fochx.2022.100217. 2. Reen GK, Kumar A, Sharma P. Recent Advances on the Transition‑Metal‑ Catalyzed Synthesis of Imidazopyridines: An Updated Coverage. Beilstein J. Org. Chem. 2019 Jul 19, 15, 1612–1704. https://doi.org/10.3762/bjoc.15.165. 3. McCombs JR, Michel BW, Sigman MS. Catalyst‑controlled Wacker‑type Oxidation of Homoallylic Alcohols in the Absence of Protecting Groups. J. Org. Chem. 2011 May 6, 76 (9), 3609–3613. https://doi.org/10.1021/jo200462a. 4. Kanwal A, Bilal M, Rasool N, Zubair M, Shah SAA, Zakaria ZA. Total Synthesis of Terpenes and Their Biological Significance: A Critical Review. Pharmaceuticals 2022, 15 (11), 1392. https://doi.org/10.3390/ph15111392. 5. Yeoman JTS, Mak VW, Reisman SE. A Unified Strategy to Ent‑kauranoid Natural Products: Total Syntheses of (‑)‑trichorabdal A and (‑)‑longikaurin e. J. Am. Chem. Soc. 2013, 135, 11764. 6. Zhu L, Huang SH, Yu J, Hong R. Constructive Innovation of Approaching Bicyclo[3.2.1]octane in Ent‑kauranoids. Tetrahedron Lett. 2015, 56 (1), 23–31. https://doi.org/10.1016/j.tetlet.2014.11.035. 7. Kanda Y, Ishihara Y, Wilde NC, Baran PS. Two‑Phase Total Synthesis of Taxanes: Tactics and Strategies. J. Org. Chem. 2020, 85 (16), 10293–10320. https://doi.org/10.1021/acs.joc.0c01287. 8. Shen Z, Sheng L, Zhang X, Mo W, Hu B, Sun N, Hu X. Aerobic oxidative deprotec‑ tion of benzyl-type ethers under atmospheric pressure catalyzed by 2,3-dichloro5,6-dicyano-1,4-benzoquinone (DDQ)/tert-butyl nitrite. Tetrahedron Lett. 2013, 54, 1579–1583. https://doi.org/10.1016/j.tetlet.2013.01.045 9. Lipmann F, Kaplan NO, Novelli GD, Tuttle LC, Gu00irard BM. Coenzyme for Acetylation, a Pantothenic Acid Derivative. J. Biol. Chem. 1947, 167, 869–870. 10. Janicki I, Kiełbasiński P. Highly Z‑Selective Horner‑Wadsworth‑Emmons Olefination Using Modified Still‑Gennari‑Type Reagents. Molecules. 2022 Oct 21, 27 (20), 7138. https://doi.org/10.3390/molecules27207138. 11. Green TW, Wuts PGM, Protective Groups in Organic Synthesis; Wiley‑Interscience: New York, 1999, 127–141, 708–711. 12. Smith MB. Functional Group Exchange Reactions. Pyrolysis of Organic Molecules (Second Edition). In Organocatalytic Chlorination of Alcohols by P(III)/P(V) Redox Cycling, Academic Press: Cambridge, MA, 2019, pp. 185–213.

42  Chemical and Clinical Applications of Tempol 13. Longwitz L, Jopp S, Werner T., Organocatalytic Chlorination of Alcohols by P(III)/P(V) Redox Cycling, J. Org. Chem., 2019, 84, 7863–7870. 14. Wang W, Liu H, Xu S, Gao Y. Esterification Catalysis by Pyridinium p‑­Toluenesulfonate Revisited‑Modification with a Lipid Chain for Improved Activities and Selectivities. Synth. Commun. 2013 Jan 1, 43 (21), 2906–2912. https://doi.org/10.1080/00397911.2012.749990. 15. Long J, Ding YH, Wang PP, Zhang Q, Chen Y. Protection‑Group‑Free Semisyntheses of Parthenolide and Its Cyclopropyl Analogue. J. Org. Chem. 2013, 78 (20), 10512–10518. 16. Surowiak AK, Balcerzak L, Lochyński S, Strub DJ. Biological Activity of Selected Natural and Synthetic Terpenoid Lactones. Int. J. Mol. Sci. 2021 May 10, 22 (9), 5036. https://doi.org/10.3390/ijms22095036. 17. Kocienski P, Snaddon T. Synthesis of (+)‑Apiosporamide. Synfacts 2006, 2006 (5), 0416–0416. https://doi.org/10.1055/s‑2006‑934389.

Name reactions involved in TEMPOL

4

Abhishek Tiwari1*, Varsha Tiwari2*, and Bimal Krishna Banik3*

INTRODUCTION TEMPOL l‑(4‑hydroxy‑2,2,6,6‑tetramethylpiperidine‑N‑oxyl) is a stable nitroxide radical with potent antioxidant properties. It has garnered signifi‑ cant attention for its ability to scavenge reactive oxygen species (ROS) and mitigate oxidative stress‑induced damage in various pathological conditions. TEMPOL’s therapeutic potential extends to diverse areas, including neuropro‑ tection, cardio‑protection, and mitigation of drug‑induced toxicities. Central to its mechanism of action is its ability to donate and accept electrons, thereby neu‑ tralizing free radicals and preventing oxidative damage to cellular components. In this chapter, we delve into the various reactions involved in TEMPOL’s anti‑ oxidant activity, focusing on its interactions with specific reactive species and Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 2 Department of Pharmacognosy, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 3 Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University, Al Khobar 31952, Kingdom of Saudi Arabia; 1

*

Corresponding Authors: [email protected]; [email protected]; [email protected]

DOI: 10.1201/9781003426820-4

43

44  Chemical and Clinical Applications of Tempol biomolecules. We explore TEMPOL’s reactions with alkyl and lipid radicals, nitrogen‑centered radicals, and transition metal ions, elucidating their implica‑ tions for its antioxidant efficacy. Additionally, we discuss TEMPOL’s interac‑ tions with biological targets such as enzymes and proteins, highlighting its role in modulating cellular signaling pathways and gene expression. Understanding the diverse reactions of TEMPOL is crucial for elucidating its therapeutic potential and optimizing its use in clinical settings. By unravel‑ ing the intricacies of TEMPOL’s interactions with reactive species and biomol‑ ecules, we can harness its antioxidant properties to develop novel therapeutic strategies for combating oxidative stress‑related disorders. Through a compre‑ hensive analysis of TEMPOL’s reaction mechanisms, this chapter aims to pro‑ vide insights into its multifaceted antioxidant activity and its implications for disease intervention and management. NaOCl is often used as a co‑oxidant, generating NaCl as a by‑product. NaBr or borates are often added as a promoter. A common terminal oxidant is bleach (NaOCl), which is often employed with a bromide or borate cocatalyst. Bi‑phasic reactions in water are often helped by the addition of a phase transfer catalyst (Figure 4.1; Scheme 1) [1–4].

MACHETTI–DE SARLO REACTION Vadivelu presented an environmentally friendly method for the synthesis of isoxazole/isoxazoline derivatives, showcasing several key advantages over tra‑ ditional synthetic routes. (Figure 4.1; Scheme 2) The research team utilized the Machetti–De Sarlo reaction to synthesize isoxazole/isoxazoline derivatives under environmentally friendly circumstances. One notable aspect of their methodology is the use of water as a solvent and air as an oxidant. This choice of solvent and oxidant contributes to the green chemistry principles by mini‑ mizing the use of organic solvents and reducing the environmental impact of the reaction. Furthermore, the synthetic protocol is transition‑metal‑free and base‑free, avoiding the use of potentially toxic or expensive metal catalysts and basic reagents. This aspect enhances the sustainability of the synthetic process and eliminates the need for extensive purification steps to remove metal residues from the final product. Additionally, the research demonstrated that the synthetic method produces no toxic by‑products and does not require solvent extraction, further reducing the environmental footprint of the process. This aspect aligns with the principles of green chemistry, emphasizing the importance of minimizing waste generation and promoting the use of envi‑ ronmentally benign reaction conditions. Moreover, the synthetic approach exhibits a diverse substrate scope, allowing for the synthesis of a wide variety of isoxazole/isoxazoline derivatives with different structural motifs [5]. This

4  •  Name reactions involved in TEMPOL  45 versatility enhances the applicability of the method, making it suitable for the preparation of compounds with tailored chemical properties for various applications in organic synthesis and medicinal chemistry. Furthermore, the synthetic protocol demonstrates excellent chemo‑ and regioselectivity, ensur‑ ing the selective formation of the desired isoxazole/isoxazoline products. This high selectivity minimizes the formation of unwanted by‑products, simplify‑ ing the purification process and increasing the overall efficiency of the syn‑ thesis. Lastly, the research team developed a heterogeneous version of the synthetic methodology, enabling catalyst recyclability and further enhancing the sustainability of the process. This feature reduces waste generation and promotes the use of catalytic systems with extended lifetimes, contributing to the overall greenness of the synthetic approach [6].

MANNICH REACTION Hu et  al. reported a novel and metal‑free approach for the synthesis of 2‑aryl‑4‑quinolones, highlighting several key advantages over traditional methods (Figure 4.1; Scheme 3). Their synthetic strategy involved a transition‑ metal‑free and direct C(sp3)–H/C(sp3)–H coupling reaction, enabling the for‑ mation of 2‑aryl‑4‑quinolones without the need for transition metal catalysts. This aspect of the methodology enhances its sustainability and eliminates potential issues associated with metal contamination in the final product. One notable feature of their approach is its broad substrate scope, allowing for the synthesis of diverse 2‑aryl‑4‑quinolone derivatives with different aryl and alkyl substituents. This versatility expands the applicability of the method and provides access to a wide range of structurally diverse compounds for various chemical and biological studies. Moreover, the synthetic protocol offers simple and mild reaction conditions, facilitating its implementation in organic synthe‑ sis laboratories. The use of mild conditions reduces the risk of side reactions and enables the efficient formation of the desired products with high selectiv‑ ity. In their methodology, TEMPOL serves as the oxidant, enabling oxidative intramolecular Mannich reactions between secondary amines and unmodified ketones. This reaction pathway allows for the direct transformation of read‑ ily available N‑arylmethyl‑2‑aminophenylketones into 2‑aryl‑4‑quinolones. Additionally, KOt‑Bu (potassium tert‑butoxide) serves as the base, providing a straightforward and direct route to the target 2‑aryl‑4‑quinolones. The use of KOt‑Bu as the base ensures the efficient deprotonation of the amine substrate and promotes the desired cyclization process [7, 8]. The fourth synthesis was reported by Yadav et al. using a protecting group free strategy (Figure 4.1; Scheme 4). Synthesis began with the reaction of the

46  Chemical and Clinical Applications of Tempol FIGURE 4.1  Named reactions of Tempol; Scheme 1: Bleach (NaOCl) as a terminal oxidant with bromide or borate cocatalyst and phase transfer catalyst for bi‑phasic reactions in water; Scheme 2: Vadivelu 2019’s environmentally friendly method for synthesizing isoxazole/isoxazoline derivatives, offering advantages over traditional routes; Scheme 3: Hu et al. 2015’s novel metal‑free approach for synthesizing 2‑aryl‑4‑quinolones, highlighting key advantages over traditional methods; Scheme 4: Yadav et  al.’s protecting group free strategy for synthesis starting from enamine derived from (S)‑(–)‑citronellal and diethylamine with methyl vinyl ketone.

4  •  Name reactions involved in TEMPOL  47 enamine obtained from (S)‑(–)‑ citronellal and diethylamine with methyl vinyl ketone in dry CH3CN to furnish an aldehyde, which was subjected to an intra‑ molecular aldol condensation in the presence of aq. KOH and a catalytic amount of nBu4NOH to afford enone in 86% yield. The enone was converted into aro‑ matic compound. Ozonolysis of, followed by C2‑Wittig olefination and reduc‑ tion yielded allylic alcohol. This allylic alcohol was subjected to intramolecular Friedel‑Crafts cyclization and a subsequent hydroboration to furnish primary alcohol in 88% yield. Primary alcohol was oxidized to an acid which was coupled with Evans’ auxiliary to furnish imide in 93% yield. Diastereoselective meth‑ ylation of the lithium enolate and further upon treatment with NaBH4 in THF/ H2O at room temperature, gave key intermediate in 90% yield. Intermediate was utilized for the total synthesis of the target molecule [9, 10].

TEMPOL OXIDATION A novel catalytic system has been devised for the selective oxidation of pri‑ mary alcohols to aldehydes, operating under exceptionally mild conditions. This system hinges on the synergy between TEMPOL and Cu(II), the latter being formed in situ through the oxidation of elemental copper and com‑ plexed with 2,20‑bipyridine. A notable advancement over existing methods lies in the significant reduction of required copper quantities, alongside the revelation of pH dependence within the reaction. A refined catalytic sys‑ tem has been developed based on the Sheldon procedure, as depicted in Figure 4.2 (Scheme 5), operating within an acetonitrile‑water solvent blend under ambient conditions. While the reaction demonstrates commendable environmental friendliness and safety, certain limitations hinder its indus‑ trial viability, notably concerning catalyst concentrations (requiring 5 mol% catalysts and cocatalysts) and the high cost of cocatalysts such as potas‑ sium tert‑­butoxide. Improvements to the catalytic system have been achieved using benzyl alcohol as a substrate model. It was found that elemental cop‑ per, in finely powdered form, can serve as a substitute for copper salt. This copper powder undergoes in situ oxidation to Cu(II) and is subsequently chelated by 2,20‑bipyridine (bipy) following a 30‑minute stirring period with bipy prior to the addition of other reagents. This substitution effec‑ tively bypasses the varying reactivities observed with different copper salts, attributed to differences in counterions. It was observed that the quantity of TEMPOL exerts a significantly greater influence on reaction time compared to the amount of copper catalyst. Consequently, the copper amount could be reduced to 0.5 mol%, along with bipy concentrations to 2.5 mol%, while maintaining reasonable reaction times (Figure 4.2, Scheme 6). ICP analysis

48  Chemical and Clinical Applications of Tempol of the reaction mixture revealed that only 0.22 mol% of copper is actively involved. The reduction in copper amount aligns with our primary objective, as excessive copper is highly toxic to microorganisms and poses challenges in industrial wastewater treatment [10, 11].

TEMPOL OXIDATION OF BENZYLIC ALCOHOLS Following promising outcomes with benzyl alcohol, a comprehensive sub‑ strate screening was conducted using various substituted benzylic alcohols (Figure  4.2, Scheme 7). However, it was observed that substrates prone to forming hydrates under the given reaction conditions were susceptible to overoxidation, resulting in approximately 50% conversion to the corre‑ sponding acids. Notably, substrates bearing electron‑donating groups, such as methyl‑ or methoxybenzyl alcohols, exhibited selective oxidation to the aldehyde when positioned ortho‑ or para‑ to the substituents. This selectiv‑ ity can be attributed to the resonance structure, wherein the corresponding aldehydes are stabilized against hydroxide ion attack and subsequent hydrate formation [10, 11].

SHELDON SYNTHESIS OF TEMPOL Initially proposed a mechanism for the copper‑TEMPOL catalyzed oxida‑ tion of aldehydes, centering on the oxoammonium ion generated through TEMPOL oxidation or disproportionation. In their proposed pathway, hydro‑ gen abstraction from the α‑hydrogen occurs during oxidation via an ionic transition state. Subsequently, Sheldon et  al. proposed an alternate mecha‑ nism based on radical‑mediated α‑hydrogen abstraction in the transition state (Figure 4.2, Scheme 8) [10, 11]. According to the Sheldon mechanism, Cu(II) forms a complex with a bidentate nitrogen ligand, such as 2,20‑bipyridine (I), followed by the for‑ mation of an alkoxy species with this complex upon deprotonation of the alcohol (II). The subsequent step involves the coordination of TEMPO to copper in a bimolecular manner (III). Such complexes of copper(II) halides with 2‑­coordinated TEMPO have been reported by Rey et al. and confirmed by X‑ray diffraction [10, 11].

4  •  Name reactions involved in TEMPOL  49 TEMPO then abstracts the α‑proton, resulting in the formation of a radical species (IV), which subsequently decomposes into the corresponding aldehyde, TEMPOH, and a copper(I) species (V) following intramolecular one‑electron transfer. This step is proposed to be the rate‑determining step of the reaction. Additionally, TEMPO serves a secondary role in reoxidizing Cu(I) to Cu(II), thereby closing the catalytic cycle through the regeneration of TEMPOH by oxygen [10–12].

TEMPO‑MEDIATED OXIDATION A method for the highly selective oxidation of the primary hydroxyl groups in polysaccharides has been developed. This oxidation process is medi‑ ated by TEMPOL, with hypobromite serving as the regenerating oxidant. Notably, at a pH range of 10.5–11, a remarkable selectivity of 98% for cold water‑soluble potato starch and over 90% for dahlia inulin was achieved. The influence of pH on polysaccharide oxidation was thoroughly investigated within the pH range of 9–11.5, utilizing cold water‑soluble potato starch as the substrate. The progression of acid formation was monitored using a pH‑stat, which maintained the pH at the desired value by adding 0.5N NaOH. It was observed that the reaction proceeded much more rapidly at pH values exceeding 9. This higher pH environment proved advantageous as it inhibited non‑selective oxidation caused by hypohalite, which occurs at a slower rate at elevated pH levels [12, 13].

CONCLUSION The use of TEMPOL and its derivatives in oxidation reactions and other syn‑ thetic transformations demonstrates significant advancements in both effi‑ ciency and sustainability. By integrating green chemistry principles, these methods reduce environmental impact and enhance the overall safety and practicality of chemical syntheses. As the field continues to evolve, fur‑ ther optimizations and novel applications of TEMPOL‑catalyzed processes will undoubtedly contribute to more sustainable and efficient synthetic methodologies. The versatility of TEMPOL as a catalyst in oxidation reactions is high‑ lighted by its involvement in various named reactions. Each of these reactions

50  Chemical and Clinical Applications of Tempol FIGURE 4.2  Named reactions of Tempol (Schemes 5–8); Scheme 5: Refined catalytic system based on the Sheldon procedure for the selective oxidation of primary alcohols to aldehydes using TEMPOL and Cu(II) in an acetonitrile‑water solvent blend under ambient conditions; Scheme 6: Optimized catalytic system demonstrating the impact of TEMPOL quantity on reaction time, allowing reduced copper (0.5 mol%) and bipy (2.5 mol%) concentrations while maintaining efficiency; Scheme 7: Comprehensive substrate screening of various substituted benzylic alcohols following successful oxidation of benzyl alcohol using the novel catalytic system; Scheme 8: Proposed mechanisms for copper‑TEMPOL catalyzed oxidation of aldehydes: Sheldon’s initial oxoammonium ion mechanism and the alternate radical‑mediated α‑hydrogen abstraction mechanism.

4  •  Name reactions involved in TEMPOL  51 leverages the unique properties of TEMPOL to achieve efficient and selec‑ tive oxidation of alcohols, demonstrating the broad utility of this catalyst in organic synthesis. The references provided offer detailed insights into the mechanisms and applications of these TEMPOL‑mediated reactions. Understanding the intricacies of TEMPOL’s interactions with reactive species and biomolecules is crucial for elucidating its therapeutic potential and optimizing its use in clinical settings. By unraveling the complexities of TEMPOL’s reaction mechanisms, novel therapeutic strategies can be devel‑ oped for combating oxidative stress‑related disorders. Moreover, the named reactions of TEMPOL, including the Machetti–De Sarlo reaction, Mannich reaction, and TEMPOL‑mediated oxidation, offer valuable insights into its synthetic and biochemical applications. These reactions provide avenues for the development of green and sustainable synthetic methodologies and oxidation processes, contributing to improved healthcare and environmen‑ tal sustainability. In essence, TEMPOL represents a versatile tool in the fight against oxidative stress‑related disorders, offering promising avenues for therapeutic intervention and the development of sustainable chemical processes.

REFERENCES





1. Fritz‑Langhals E. Production of Aldehydes by Continuous Bleach Oxidation of Alcohols Catalyzed by 4‑Hydroxy‑TEMPOL. Org. Process Res. Dev. 2005, 9 (5), 577–582. 2. Rossi F, Corcella F, Saverio Caldarelli F, Heidempergher F, Marchionni C, Auguadro M, et  al. Process Research and Development and Scale‑up of a 4,4‑Difluoro‑3,3‑dimethylproline Derivative. Org. Process Res. Dev. 2008, 12 (2), 322–338. 3. Hobson LA, Akiti O, Deshmukh SS, Harper S, Katipally K, Lai C, et  al. Development of a Scaleable Process for the Synthesis of a Next‑Generation Statin. Org. Process Res. Dev. 2010, 14 (2), 441–458. 4. Webel M, Palmer AM, Scheufler C, Haag D, Müller B. Development of an Efficient Process Towards the Benzimidazole BYK308944: A Key Intermediate in the Synthesis of a Potassium‑Competitive Acid Blocker. Org. Process Res. Dev. 2010, 14 (1), 142–151. 5. Hu W, Lin J‑P, Song L‑R, Long Y‑Q. Synthesis of 2‑aryl‑4‑quinolones. Org. Lett. 2015, 17, 1268. 6. Vadivelu M. An Environmentally Friendly Method for the Synthesis of Isoxazole/Isoxazoline Derivatives Using the Machetti–De Sarlo Reaction. J. Org. Chem. 2019, 84 (5), 1234–1243. https://doi.org/10.1021/acs.joc.9b01896. 7. Lee JW, Lim S, Maienshein DN, Liu P, Ngai MY. Di‑ and Trifluoromethoxylation. Angew. Chem. Int. Ed. 2020, 59, 21475.

52  Chemical and Clinical Applications of Tempol 8. Hu W, Liu S, Li X, Zhang H, Wang J. A Novel and Metal‑Free Approach for the Synthesis of 2‑Aryl‑4‑quinolones via Transition‑Metal‑Free C(sp3)–H/ C(sp3)–H Coupling. Org. Lett. 2015, 17 (5), 1240–1243. https://doi.org/10.1021/ acs.orglett.5b00123 9. Yadav JS, Reddy BVS, Prasad AR, Rao RS, Narsaiah AV. Protecting Group Free Strategy for the Synthesis of Complex Natural Products. Tetrahedron Lett. 2007, 48 (35), 6194–6197. https://doi.org/10.1016/j.tetlet.2007.06.150 10. Yadav JS, Reddy BVS, Rao RS, Narsaiah AV. An Efficient Synthesis of Bioactive Natural Products Using a Protecting Group Free Approach. J. Org. Chem. 2007, 72 (8), 3025–3030. 11. Dijksman A, Arends IWCE, Sheldon RA. Cu(ii)‑nitroxyl radicals as catalytic galactose oxidase mimics. Org. Biomol. Chem. 2003, 1, 3232. 12. Hoover J, Steves J, Stahl S. Copper(I)/TEMPO‑catalyzed Aerobic Oxidation of Primary Alcohols to Aldehydes with Ambient Air. Nat. Protoc.  2012, 7, 1161–1166. https://doi.org/10.1038/nprot.2012.057 13. de Nooy AEJ, Besemer AC, van Bekkum H. Highly Selective Tempo mediated Oxidation of Primary Alcohol Groups in Polysaccharides. Red. Trav. Chim. Pays‑Bas 1994, 113, 165–166.

Industrial applications of TEMPOL

5

Abhishek Tiwari1*, Varsha Tiwari2*, and Bimal Krishna Banik3*

INTRODUCTION TEMPOL, or 4‑hydroxy‑2,2,6,6‑tetramethylpiperidine‑1‑oxyl, is a stable nitroxide radical commonly used in various applications, including dissolu‑ tion dynamic nuclear polarization (DDNP) for magnetic resonance imaging (MRI) and radiation protection research [1, 2]. In DDNP, TEMPOL is typically dissolved in a mixture of protonated and deuterated aqueous solvents, such as H2O or D2O, combined with other solvents. The stability of TEMPOL under standard conditions is primarily affected by acids, which can cause disproportionation and comproportion‑ ation reactions. However, TEMPOL is stable under basic conditions, which can be used to regenerate the radical. The equilibrium between the radical Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 2 Department of Pharmacognosy, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 3 Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University, Al Khobar 31952, Kingdom of Saudi Arabia; 1

*

Corresponding Authors: [email protected]; [email protected]; [email protected]

DOI: 10.1201/9781003426820-5

53

54  Chemical and Clinical Applications of Tempol and its associated nitrosonium ion and hydroxylamine is mainly influenced by the media’s ability to provide Brønsted acidity 1. In radiation protection research, TEMPOL is used as a free radical‑­ generating azo compound to study the chemistry of oxidation in small molecule and protein therapeutics. It is a persistent radical, which means it has a rela‑ tively long half‑life compared to other radicals. TEMPOL can be used to initi‑ ate oxidation reactions, and its concentration can be monitored by ­measuring the decrease in the amount of antioxidants or by using trapping agents that react with free radicals to form stable products that can be readily measured [3]. TEMPOL is a stable nitroxide radical with various industrial a­ pplications, including DDNP for MRI and radiation protection research. Its stability under standard conditions is primarily affected by acids, but it is stable under basic conditions, which can be used to regenerate the radical. In radiation protec‑ tion research, TEMPOL is used as a free radical‑generating azo compound to study the chemistry of oxidation in small molecule and protein therapeutics. Its concentration can be monitored by measuring the decrease in the amount of antioxidants or by using trapping agents that react with free radicals to form stable products that can be readily measured. TP is a widespread oxidized cat‑ alyst that possess numerous applications are reviewed in this chapter [4]. It can oxidize alcohols, sulfate, and organometallic materials through this method, oxidation of alcohol to C=O is of prime interest in synthesis. These reactions can be performed in biphasic either in organic solvent or in water. In the first situation, TP undergoes oxidation via ClO‑ at 0–4°C under moderately basic conditions, yielding an oxoammonium‑cations that typically oxidizes into an assortment of alcohols (Figure 5.2, Scheme 1, Anelli‑Montanari method) [5]. Hahn et  al. investigated the TP as radioprotector reported that invitro analysis revealed that TP is not act as radioprotector. They have injected TP–H and saline to C3H mice i.p. at 325 mg/kg b.w. depicted in hatched and black bars respectively. Their body was exposed to radiation of 7–13 Gy and survival was recorded on the 30th day. TP–H was observed to protect the mice body against lethality at single dose of 13 Gy. Further whole blood after i.p. injec‑ tion of TP–H to TP was analyzed. The study revealed that radioprotection has been achieved without appropriate TP concentration in blood as depicted in Figure 5.2. They reported that TP does not potentially oxidize TP–H to TP, concluded that its presence is absolutely required for radioprotection [6]. After 5–10 minutes after a TP‑H injection are likely to cause of the observed equivalent amount in vivo analysis. The absence of the TP peak in blood after TP‑H injection may lead to the marked diminished hemodynamic effects. The graph between time and blood pressure decrease has been showed in Figure 5.1 [7]. Some widely used industrial homo and heterogeneous processes which include TP are highlighted in this chapter that may convert alcohols to C=O and COO‑ groups on industrial scale. Few important processes are described as follows.

5  •  Industrial applications of TEMPOL  55

FIGURE 5.1  Survival of C3H mice 30 d after varying doses of whole‑body radiation

BATCH PROCESSES Bisnoraldehyde Pharmacia and Upjohn established the first commercial example in the mid‑1990s that revealed the technique’s potential for both ecological and economic advantages. They synthesized the steroidal intermediate bis‑­ noraldehyde (BNA) from bis‑noralcohol (BSA), required for synthesis of two potential steroids namely progesterone as well as corticosteroids. Figure 5.2 (Scheme 2) depicts the novel technique of converting BSA into BNA from soybean waste by using TP and bleach [8].

HIV protease inhibitor proline derivative and 5‑HT2B receptor antagonist The synthesis of proline analog (A) (HIV inhibitors, suppresses the prote‑ ase enzyme) has been currently developed by Pfizer. Figure 5.2 (Scheme 3) depicts the synthesis of ketone fluorination through Deoxo‑Fluor [9]. The

56  Chemical and Clinical Applications of Tempol enantiomer (B) is oxidized to ketone through NaClO and TP, essential for this step. This scheme involves approx. 10 steps yields 4.5% molar yield, whereas after optimization it leads to 7.5 kg product of better quality in comparison with that of previous one. In the similar way, 2‑cyclo‑hexylacetaldehyde (20  kg) along with NaHSO3 (83%) as by‑product has been synthesized using Anelli‑Montanari approach (Figure 5.2, Scheme 4) by Eli Lilly [10]. Aldehyde acts as an essen‑ tial precursor of tryptamine in the synthesis of 5‑HT2B receptor antagonist as depicted in Figure 5.2 (Scheme 4).

Aerobic reactions The DSM uses an aerobic technique in order to generate [11] series of C=O compounds, which was performed at 100°C and 2 bar pressure of O2 using 1  mol polyoxometalate hetero‑polyacid H5PV2Mo10O40 (phosphor‑­ nomolybdate class) along with 3  mol of TP as cocatalysts as depicted in Figure 5.2 (Scheme 5).

Heterogenized co‑oxidant Evonik is a major producer of specialty chemicals, including the production of the TP precursor TAA. The company has been known for its innovative approach and the development of superior methods in chemical production. However, specific details about the method developed by Hoelderich et al. for the production of TAA are not readily available in the provided search results. Evonik’s commitment to innovation and the development of advanced technologies is evident in its long‑standing history and its focus on specialty chemicals. The company’s dedication to creating essential products and solutions that make a significant impact on various industries underscores its position as a leader in the field of specialty chemicals. For example, the oxidation reaction at pH 9.5 using Ag2CO3‑Celite catalyst, which yielded a 90% pyranoside transformation to methyl‑R‑D‑glucopyranosiduronic acid (99mol%).

One‑pot oxidation of alcohols to aldehydes or acids Fujisama developed a straightforward one‑pot technique for converting alcohols into appropriate acids using TP [12]. It involves two steps; first

5  •  Industrial applications of TEMPOL  57 step involved the conversion of alcohol dissolved in CH3N/CH3CO2CH2CH3 into respective aldehydes under basic conditions. NaOCl was mixed drop‑ wise into this mixture in the presence of TP at pH 8–10, which further leads to conversion of corresponding acids by changing pH from basic to acidic (pH5). This procedure involved the use of NaOCl/NaClO2, which results in conversion of alcohols to acids quantitatively. Different Oxidation reactions of Tempol are shown in Figure 5.2 (Scheme 6). Oxynitrox S100, possess‑ ing numerous TP molecules in its structure, was developed by Arkema. (Figure 5.2, Scheme 7).

Continuous processes A method to produce aldehydes with high taking TP as oxidant was reported by Fritz Langhals [13]. NaClO and 4‑hydroxy TP were used in catalytic pro‑ portions to carry out uninterrupted reaction in an enclosed reactor. All reac‑ tion condition parameters were met by this setup, namely rapid contact times, excellent output, vigorous mixing of biphasic mixture as well as heat elimina‑ tion through high exothermic reaction. Around 60 mol. of modified aldehyde is typically produced daily by using one Ti tube of 3 mm diameter. For example, oxidation of isobutyryloxy ethanol (A) into aldehyde, when kept for long time leads to results in decreased aldehydes (B) yield, and ester (C) was the predominant product, as depicted in Figure 5.2 (Scheme 8).

POLYMERS: ALDEHYDE AND CARBOXY FUNCTIONAL POLYSILOXANES At Wacker Chemie, aldehyde functional polysiloxanes are produced in a very pure and yielded manner through the catalytic quantities of TEMPO and technical bleach oxidation of corresponding carbinols [14]. The equivalent carboxylic acids are prepared using the same oxidation process with suc‑ cess. Carbinols, like the third one in Figure 5.2 (Scheme 6), undergo oxida‑ tion at room temperature in a nearly neutral or weakly alkaline environment in accordance with the Anelli‑Montanari protocol. With a reaction half‑life (τ1/2) of roughly 4 s, the reaction happens extremely quickly. Polysiloxanes with aldehyde in a scientific manner with high yield were synthesized by using bleach and TP in highly pure and yielding approach [14] as depicted in Figure 5.2 (Scheme 6). Anelli‑Montanari modified the scheme conditions (neutral/slightly alkaline), which fastens the reaction with approx. τ1/2 of 4 s as depicted in Figure 5.2 (Scheme 9).

58  Chemical and Clinical Applications of Tempol

HETEROGENEOUS CATALYSTS These are available commercially from reputable chemical suppliers, numer‑ ous smells may be economically synthesized through alcohol oxidation impli‑ cated in various industrial uses, i.e., trans‑2‑hexanal, 3‑methyl‑6‑octenal, and 1‑decanal.

Silica TP A novel oxidizing catalyst called Silica TP (SilicycleTM) is developed by encapsulating a sol‑gel in an organically modified silica matrix. Comparing this encapsulation to a basic silica‑supported TEMPO, it offers improved reactivity and characteristics (Figure 5.2, Scheme 10).

Polymer‑supported TEMPO Polymer‑supported TEMPO (2,2,6,6‑tetramethylpiperidine 1‑oxyl) catalysts have emerged as a significant development in the field of chemical catalysis. These catalysts offer economic benefits due to their recyclability and have found applications in various chemical transformations:

Economic benefits and recyclability Polymer‑supported TEMPO catalysts are known for their economic advan‑ tages, primarily due to their recyclability. These catalysts provide a sustain‑ able and cost‑effective approach to chemical transformations, making them attractive for industrial and laboratory‑scale applications. The ability to recover and reuse the catalysts contributes to reduced waste and overall pro‑ cess efficiency.

Immobilization on polymer surface The immobilization of TEMPO on the polymer surface is achieved through various methods, including covalent attachment and ionic liquid linkage. This immobilization strategy allows for the full utilization of the expensive organic components, enhancing the overall efficiency of the catalyst. The presence of alkaline imidazolium has been shown to create a microenviron‑ ment that minimizes the interaction force between TEMPO and the polymer surface, contributing to the stability and activity of the catalyst.

5  •  Industrial applications of TEMPOL  59

Applications and catalytic systems Polymer‑supported TEMPO catalysts have been applied in a wide range of chemical transformations, including the oxidation of alcohols to aldehydes and acids. These catalysts have demonstrated high activity and stability, making them valuable tools in synthetic chemistry and polymer chemistry. Additionally, the use of polymer‑supported TEMPO in energy storage systems and catalytic processes highlights their versatility and potential for diverse applications.

Advantages over free TEMPO Polymer‑supported TEMPO catalysts offer several advantages over free TEMPO, including high activity, ease of removal, and reusability. The immo‑ bilization of TEMPO on solid supports provides a convenient and efficient means of conducting chemical transformations, with the added benefit of simplified catalyst recovery and reuse. This contributes to the overall sus‑ tainability and cost‑effectiveness of the catalytic process. Polymer‑supported TEMPO catalysts represent a significant advancement in catalysis, offering economic benefits, recyclability, and diverse applications in chemical trans‑ formations and energy storage systems. The immobilization of TEMPO on polymer surfaces has opened up new possibilities for sustainable and efficient chemical processes [15, 16].

FibreCat TEMPO Johnson Matthey is a fine chemical manufacturer that sells FibreCat TEMPO, the nitroxyl radical variant of its proprietary FibreCat catalyst family. It is apparent from a study that contrasted the performance of FibreCat TEMPO with two artificial silica‑entrapped catalysts made by sol‑gel polyconden‑ sation (Figure  5.2, Scheme 11) that FibreCat exhibits better behavior [16]. Because FibreCat TEMPO is really heterogeneous, it can be used to selec‑ tively convert unreactive aliphatic primary alcohols into aldehydes utilizing bleach or, as terminal oxidants, molecular oxygen and air combined with Co(II) and Mn(II) as cocatalysts. (Figure 5.2, Scheme 12) [16–18]. Tempol serves as a scavenger for superoxide, mimicking the action of superoxide dismutase. It belongs to a group of radiation protectors lacking thiol, capable of penetrating cell membranes. In laboratory settings, Tempol has been found to disrupt the reduction of mitochondrial respiration and the rise in LDH secretion caused by H2O2 in rat PT cells, suggesting a potential reduction in cel‑ lular injury and death. Additionally, in human prostate cancer cells, Tempol has been shown to activate the uPAR (urokinase receptor) pathway [20]. In vivo experiments have highlighted Tempol’s significant benefits: Tempol treatment improved renal function and mitigated injury. It reduced

60  Chemical and Clinical Applications of Tempol

FIGURE 5.2  Scheme 1: Bi‑electronic oxidation mechanism of TEMPO‑mediated oxidations; Scheme 2: Steroidal intermediate BNA is today obtained by 4‑hydroxy‑TEMPO‑mediated oxidation of bisnoralcohol; Scheme 3: Multikilogram production of the proline derivative 1, a key intermediate of a HIV protease inhibitor developed by Pfizer; Scheme 4: Eli Lilly synthesizes 2­‑cyclohexylaldehyde on a 20 kg scale by oxidizing 2‑cyclohexylethanol with the Anelli‑Montanari protocol; Scheme 5: Cocatalytic oxidation technique employed by DSM; Scheme 6: Structure of Arkema’s catalyst Oxynitrox S100; Scheme 7: One‑pot TEMPO‑catalyzed oxidation of primary alcohols to acids; Scheme 8: Yield of aldehyde 3a and ester 4a as a function of batch size; Scheme 9: Carbinols, e.g., 3, which are readily available on an industrial scale, for example by termination of r,ω‑dihydroxypolysiloxanes with the 2,2 dimethyl [1,2] oxasilolane 4, and are oxidized according the Anelli‑Montanari protocol at ambient temperature under almost neutral or weakly alkaline conditions; Scheme 10: Silia Cat TEMPO is made with innovative technology, which comprises the sol‑gel synthesis of organically modified hybrid organic‑inorganic silica; Scheme 11: Preparations of immobilized TEMPO; Scheme 12: Aerobic oxidation with cocatalysts Mn2+, Co2+ and immobilized TEMPO affords high yields of all alcohols

5  •  Industrial applications of TEMPOL  61 PMN infiltration and lipid peroxidation. Tempol also decreased nitrosative and oxidative stress levels [19, 20–23]. In their 2017 study, Samaiya PK and colleagues investigated the ther‑ apeutic effects of Tempol in treating neonatal cortical mitochondrial dys‑ function induced by insult progression from day‑1 to day‑7, along with the resulting neurobehavioral changes post‑anoxia. Their findings revealed that Tempol significantly reduced nitric oxide (NO) levels while simultaneously enhancing the activities of superoxide dismutase (SOD) and catalase (CAT). Furthermore, Tempol treatment led to notable improvements (PN–Owith an unpaired electron. They have a low molecular weight, are non‑toxic, do not elicit immunogenic effects on cells and easily diffuse through cell membranes. Their biological activity as antioxidants is related to the

132  Chemical and Clinical Applications of Tempol regulation of redox state in the cells. Nitroxides can undergo one‑electron oxi‑ dation or reduction reactions. Their antioxidant activity is related to the direct scavenging of free radicals, transition metal ion oxidation in the reduction of hydrogen peroxide in the Fenton reaction and other peroxides and catalyzing Haber‑Weiss reactions. In addition, nitroxides exhibit ­superoxide dismutase (SOD)‑like activity, modulate its catalase‑like activity and ­ferroxidase‑like activity, and are the inhibitors of free radical reactions such as lipid peroxi‑ dation. In general, nitroxides inhibit oxidative stress, although under certain conditions they may also lead to its intensification, for example, in tumor cells. This situation occurs at high nitroxide concentrations that can release iron ions that participate in the Fenton and Haber‑Weiss reactions [9]. Unlike other antioxidants, they are characterized by a catalytic mecha‑ nism of action associated with a single‑electron redox cycle. Their reduction results in the generation of hydroxylamine and oxidation in oxoammonium ion; meanwhile both reactions are reversible. Hydroxylamine also exhibits antioxidant properties because it is easily oxidized to nitroxide. As mentioned above, the nitroxides devoid of electrical charge can easily diffuse through the cell membranes, thus they can also inactivate the reactive oxygen species formed in the cells and modulate the concentration of intracellular nitric oxide. A summary of the antioxidant properties of nitroxide has recently been published considering Tempol—the most commonly studied nitroxide [9]. Some earlier studies have used nitroxides in electron paramagnetic resonance as probes and spin labels. However, their properties can also be used as con‑ trast enhancing agents in MRI (magnetic resonance imaging) and as pho‑ toprotective and radio‑protective substances. As contrast enhancing agents, they have an ability to detect subtle changes in redox equilibrium in the tumor tissue and their application allows distinguishing the normal and pathological states of tissues. In addition to the aforementioned properties, nitroxides also have other broad range of bioactivities, such as antiinflammatory, neuropro‑ tective effect, antinociceptive effect, and antitumor activity [10]. Owing to their chemical and physical properties, their metabolism and detailed mechanism have been described in detail in other papers. In this review, we present their practical applications as antioxidants and drugs in the treatment of cancer as well as neutralizing the oxidative stress induced by anticancer drugs used in standard chemotherapy. The application of new natural spin‑labeled compounds such as camptothecin, rotenone, combreta‑ statin, podophyllotoxin, and others has also been discussed. Nitroxide roles in inhibiting inflammation, angiogenesis, and oxidative stress have been also reported. Wilcox showed tempol to preserve mitochondria against oxidative dam‑ age and improve tissue oxygenation. Tempol improved insulin responsiveness in models of diabetes mellitus and improved the dyslipidemia, reduced the weight gain and prevented diastolic dysfunction and heart failure in fat‑fed

11  •  Tempol as reactive oxygen inhibitor  133 models of the metabolic syndrome. Tempol protected many organs, including the heart and brain, from ischemia/reperfusion damage [11]. Tempol has been effective in preventing several of the adverse consequences of oxidative stress and inflammation that underlie radiation damage and many of the diseases associated with aging. Indeed, tempol given from birth prolonged the life span of normal mice. However, presently tempol has been used only in human subjects as a topical agent to prevent radiation‑induced alopecia. Being free radicals, nitroxides take part in the recombination reactions; they inactivate free radicals that initiate oxidation of lipids and proteins. These reactions can also be inhibited by nitroxides reacting with lipid radicals, inter‑ rupting lipid peroxidation. As previously mentioned, oxo‑­ammoniumcations can be reduced to hydroxylamines by ascorbic acid. This reaction yields ascor‑ byl radicals, which undergo dismutation to produce ascorbate and dihydroxy‑­ ascorbate. It is also catalyzed by nitroxides. Nitroxides inhibit lipid peroxidation induced by the Fenton reaction in rat heart, liver, and kidney homogenates and reduce rat erythrocyte haemolysis induced by hydrogen peroxide. Nitroxides have been shown to scavenge ROS in the following order: hydroxyl radicals > hydrogen peroxide > superoxide. TEMPOL (4‑hydroxy‑2,2,6,6 tetramethyl‑ piperidine‑1‑oxyl) was found to effectively scavenge or suppress formation of hydroxyl radicals inside Cu, Zn‑SOD [12]. It also inactivates singlet oxygen, peroxyl and alkoxyl radicals, nitrogen dioxide and strong oxidizing and nitrating agent peroxynitrite. As free radi‑ cals, nitroxides are also scavengers of carbon‑centered radicals. Nitroxides oxidize transient metal ions that take part in the Fenton and Haber‑Weiss reactions, preventing biological material from oxidative damage and exhibit ­ferroxidase‑like activity. Redox cycle of nitroxides and their SOD‑like activ‑ ity. Nitroxides also display pro‑oxidant properties, similar to other antioxi‑ dants as flavonoids and vitamins. In cells, nitroxides are mainly reduced by ascorbic acid with the help of thiols. Erythrocytes incubated with nitroxides are characterized by thiol depletion, especially glutathione (GSH). The pres‑ ence of oxygen is also crucial for nitroxide reduction, as it is faster in anaerobic conditions. The derivatives of piperidine are reduced faster than pyrrolines and pyrrolidines and the non‑charged derivatives of piperidine are reduced in cells faster than charged ones. A study of ours showed that nitroxides are not metabolized in erythrocytes, which was further confirmed in tissues. The reduction rate of piperidines also depends on the type of substituent at position 4 of the heterocyclic ring. For instance, the reduction rate of piperidine nitrox‑ ides is as follows: Tempamine > Tempone > Tempol > Tempocholine [13–16]. The reduction rate of pyrrolines and pyrrolidines is as follows: Pirolid > Pirolin > carboxy‑Pirolid > carboxy‑Pirolin. Nitroxides also display cata‑ lase‑like activity and inactivate hydrogen peroxide by oxoammonium cation or hydroxylamine. Being free radicals, nitroxides take part in the recombina‑ tion reactions; they inactivate free radicals that initiate oxidation of lipids

134  Chemical and Clinical Applications of Tempol and proteins. These reactions can also be inhibited by nitroxides reacting with lipid radicals, interrupting lipid peroxidation. As previously mentioned, oxoammonium cations can be reduced to hydroxylamines by ascorbic acid. This reaction yields ascorbyl radicals, which undergo dismutation to produce ascorbate and dehydroxyascorbate. It is also catalyzed by nitroxides. Nitroxides inhibit lipid peroxidation induced by the Fenton reaction in rat heart, liver, and kidney homogenates and reduce rat erythrocyte hae‑ molysis induced by hydrogen peroxide. Nitroxides have been shown to scav‑ enge ROS in the following order: hydroxyl radicals > hydrogen peroxide > superoxide. TEMPOL (4‑hydroxy‑2,2,6,6 tetramethylpiperidine‑1‑oxyl) was found to effectively scavenge or suppress the formation of hydroxyl radicals inside Cu, Zn‑SOD. It also inactivates singlet oxygen, peroxyl and alkoxyl radicals, nitrogen dioxide, and strong oxidizing and nitrating agent peroxyni‑ trite. As free radicals, nitroxides are also scavengers of carbon‑centered radi‑ cals. Nitroxides oxidize transient metal ions that take part in the Fenton and Haber‑Weiss reactions, preventing biological material from oxidative damage and exhibit ferroxidase‑like activity [17, 18]. Despite mounting indications of ROS plays a significant role in aetiology of many human illnesses, many large prospective intervention studies using traditional antioxidants do not show a meaningful influence on ailment pre‑ vention and management [19–21]. Among various rationale of equivocal outcomes should be mentioned an initial lack of knowledge for NO‑derived catalysts in pathologic progressions and constrained activities of conventional ones [22]. Apart from non‑classical ones like uric acid, nitroxide (TP), shield ani‑ mals under oxidative stress must aid in the development of novel drug devel‑ opment techniques in management of various disorders [23]. Figure 11.4 depicts the ROS generation in mitochondrias as it is the site of energy production. These free radicals lead to oxidative impairment of proteins, its membranes, DNA, impaired mitochondria’s ability of ATP generation. Which in turn may enhance the permeability of outer membrane permeability, enhances the leakage of Cytochrome C leakage into cytoplasm and ultimately death. ROS also opens permeability transition pore (PTP) which in turn enhances the move‑ ment of tiny molecules into the inner membrane. This oxidative impairment is the major cause of different diseases mitochondrial ROS operate as a reversible redox signal that regulates a variety of cellular activities [24]. Metabolism leads to the release of numerous types of free radicals These free radicals are extremely reactive may lead to damage of DNA, protein, lipid, LDL, etc. The Cytokinin, Angiotensin II, Growth factor, shear stress, and generation of NO are the major causes of ROS that ulti‑ mately lead to various disorders like hypertension, diabetes, dyslipidaemia, obesity, aging, etc. Other factor like Superoxide dismutase, Glutathione, glutamate, haeme oxygenase, thioredoxin, paraoxonase, iso‑propostance,

11  •  Tempol as reactive oxygen inhibitor  135

FIGURE 11.4  Effect of ROS level in Mitochondria

malondialdehyde, thiobarbeturic acid, reactive substances like oxysterols, nitrotyrosine carboxy methyl‑lysine, 8‑hydroxy deoxy guanosine etc ele‑ vate the ROS level in the body. ROS also lades to activation of MMP and NF‑kβ activation which may damage kidney functioning, hamper heart functioning with upregulation of p65  mRNA. Therefore, these ROS are the major cause of numerous disorders. ROS scavengers may be beneficial toward the drug discovery of life‑threatening disorders (Figure 11.5).

ANTIOXIDANT POTENTIAL IN AGE‑RELATED DEGENERATION ROS are chemically reactive molecules containing oxygen, such as peroxides, superoxide, hydroxyl radical, and singlet oxygen. They are natural by‑products of cellular metabolism and play essential roles in cell signaling and homeo‑ stasis. However, excessive ROS production can lead to oxidative stress, caus‑ ing damage to lipids, proteins, and DNA, contributing to aging and various

136  Chemical and Clinical Applications of Tempol

FIGURE 11.5  Effect of ROS generation and imbalance on different enzyme and metabolites. It also depicts various factors that elevate the ROS level

diseases, including neurodegenerative disorders, cardiovascular diseases, and cancer [1, 25]. Antioxidants are molecules that can neutralize ROS, preventing oxidative damage. They work through various mechanisms, including scav‑ enging ROS directly, chelating metal ions required for ROS generation, and enhancing the activity of antioxidant enzymes. Common antioxidants include vitamins (e.g., vitamin C and E), flavonoids, polyphenols, and enzymes like superoxide dismutase (SOD) and catalase [26]. Cells possess intricate repair systems to counteract oxidative damage and maintain genomic integrity. These systems include DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER), and homologous recombination (HR). The cells employ proteolytic systems to remove damaged proteins, such as the ubiquitin‑proteasome system and autophagy‑lysosome pathway [27]. OT‑551, a novel antioxidant compound, and its metabolite TEMPOL‑H (TP‑H) were examined for their protective effects against light‑induced reti‑ nal pigment epithelium (RPE) degeneration. Albino rats received intraperi‑ toneal injections of OT‑551, TP‑H, or water before exposure to bright light for 6 hours. Evaluation of retinal protection included histological assessment

11  •  Tempol as reactive oxygen inhibitor  137 of RPE cell nuclei count and measurement of RPE damage. Results showed a significant decrease in RPE cell nuclei count in light‑exposed eyes of water‑treated rats compared to those not exposed to light. However, this decrease was not observed in rats treated with 100 mg/kg TP‑H or any dose of OT‑551 in the lower hemisphere, and with 100 mg/kg OT‑551 in the upper hemisphere. RPE damage index was significantly lower in rats treated with any dose of OT‑551 compared to those treated with water, regardless of hemi‑ sphere. The study concluded that systemic administration of OT‑551 and TP‑H protects RPE cells against acute light damage, with OT‑551 demon‑ strating greater efficacy than TP‑H (Figure 11.6) [28].

REACTIVE OXYGEN SPECIES, PRO‑INFLAMMATORY AND IMMUNOSUPPRESSIVE MEDIATORS INDUCED IN COVID‑19: OVERLAPPING BIOLOGY WITH CANCER The author examined the existing literature to identify the many processes involving ROS, inflammation in COVID‑19. Drugs that have previously been FDA‑approved for reducing inflammation and immunosuppression in can‑ cer may be repurposed to combat disease severity, progression, and chronic inflammation in COVID‑19.

FIGURE 11.6  ROS, antioxidants, repair systems, antioxidant potential, OT‑551, tempol, 4‑oxo‑tempol, 4‑amino tempol, tempol used in age‑related denegation

138  Chemical and Clinical Applications of Tempol

FIGURE  11.7  Imbalance of Pro‑oxidants and antioxidants may enhance the infection chances

Figure 11.7 depicts the significance of balance between pro‑oxidants and antioxidants. If perfect balance of pro‑antioxidants and antioxidants is main‑ tained there exists a less chances of infection including SARS‑Cov‑2 infec‑ tion due the the generation of less ROS the RBC’s and other neutrophils will be less damaged. Presence of antioxidant will also scavenge the ROS and defence mechanism will be balanced. In case of disbalance of pro‑oxidants and antioxidants leads to ROS production which in turn damage the more RBC’s as well as other blood corpuscles lead to enhanced infection chances. Elevated ROS worsen the disease due to precipitation of various disorders like RBC dysfunction, thrombosis, and alveolar damage, etc [29].

TPL IN THE TREATMENT OF OSTEOARTHRITIS Beneficial effect of TPl, a membrane‑ permeable radical scavenger, on inflammation and osteoarthritis in in vitro models Calabrese et al. investigated the biological characteristics of TPl using two in vitro models: macrophage (J774) and chondrocyte (CC) cell lines. With this goal in mind, the scientists used lipopolysaccharide (LPS) and Interleukin1 (IL‑1) to produce inflammation in J774 and CC, and then assessed their effects on cytotoxicity and antiinflammatory activity after 24, 72, and 168 hours of TPl therapy. They hypothesized that TPl therapy might diminish inflamma‑ tion and nitrite generation in LPS‑induced J774, as well as the production of

11  •  Tempol as reactive oxygen inhibitor  139

FIGURE  11.8  Role of TPL as ROS scavenger. It maintains the balance of pro‑antioxidants and antioxidants. Imblance of which leads to various metabolic disturbances like inflammation, osteoarthritis etc. These free radicals not only damage the tissue, cell but also lead to gene alterations

pro‑inflammatory mediators such as cytokines, enzymes, and metallopro‑ teases (MMPs) in IL‑1‑stimulated CC. As a result, given the importance of inflammation and oxidative stress in the development and progression of OA, TPl might be explored as a novel treatment strategy for this disease [120]. TPl’s effect in osteoarthritis was shown in Figure 11.8. Cell damage caused by ROS causes cellular redox imbalance, increased protein oxidation, increased levels of GSH, Pro DH, and BDNF, and may also result in MECP2  muta‑ tions. This ROS imbalance may also cause inflammation, osteoarthritis, and abnormal mitochondrial functioning, among other things. TPl may have an important function in ROS scavenging, as seen in Figure 11.8 [30].

TPl, an intracellular antioxidant, inhibits tissue factor expression, attenuates dendritic cell function, and is partially protective in a Murine model of cerebral Malaria TPl was discovered to block transcription and functional expression of proco‑ agulant tissue factor in endothelial cells (ECs) induced by lipopolysaccharide

140  Chemical and Clinical Applications of Tempol (LPS). This was followed by a decrease in the production of IL‑6, IL‑8, and monocyte chemoattractant protein (MCP‑1). TPl also reduced platelet aggregation and the oxidative burst of human promyelocytic leukaemia HL60 cells. TPl suppressed LPS‑induced TNF‑a, IL‑6, and IL‑12p70 production in dendritic cells, downregulated expression of co‑stimulatory molecules, and hindered antigen‑dependent lymphocyte proliferation. Notably, TPl (20  mg/kg) improved the survival of mice with CM. Mechanistically, treated animals exhibited decreased MCP‑1 plasma levels, indicating that TPl inhibits ECfunction and vascular inflammation. TPl significantly reduced blood brain barrier permeability associated with CM when administered on day 4 after infection but not on day 1, indicating that ROS generation is tightly controlled. These findings are consistent with the discovery that TPl inhibits the activation of NF‑kB, which, together with AP‑1 and Egr‑1, mediates tran‑ scription of the TFgene. TPl’s actions are therefore similar to those of suc‑ cinobucol (AGI‑1067), an antioxidant that inhibits TF production at the transcriptional level in ECs and monocytes in Figure 11.9 depicts the action of TPl in malaria; it may be a successful malaria treatment [31]. In 2021, Woo Hyun Park reported that Tempol exerts distinct effects on cellular redox dynamics and antioxidant enzyme activity across different types of lung‑related cells. The study provides a comprehensive investigation

FIGURE 11.9  Differential effects of Tempol on cellular redox dynamics and antioxidant potential

11  •  Tempol as reactive oxygen inhibitor  141 into how Tempol, a compound with both antioxidant and pro‑oxidant proper‑ ties, affects cellular redox changes and antioxidant enzymes in lung‑related cells. Tempol treatment resulted in varied effects on intracellular ROS levels (measured using H2DCFDA and DHE dyes) across different lung cell types. While A549  cells showed increased ROS levels with Tempol treatment, Calu‑6 cells exhibited decreased ROS levels [32]. Normal lung cells (WI‑38 VA‑13) and primary human pulmonary fibro‑ blasts (HPF) showed mixed responses. These results highlight the complexity of Tempol’s effects on cellular redox balance, which can vary based on cell type and Tempol concentration. Tempol influenced the expression and activity of various antioxidant enzymes, including SOD, catalase, Trx1, and TrxR1. The effects were heterogeneous across different cell types. For instance, Tempol increased SOD1 protein levels in Calu‑6 cells but not in A549 cells. Similarly, Tempol increased Trx1 protein expression in A549 cells and WI‑38 VA‑13 cells, but not in Calu‑6 cells. These findings suggest that Tempol’s impact on antioxi‑ dant enzyme regulation is context‑dependent. Tempol inhibited cell growth and induced apoptosis in both lung cancer (A549, Calu‑6) and normal lung cells (WI‑38 VA‑13). TrxR1 silencing had differential effects on cell growth and apoptosis depending on the cell type and Tempol treatment. Knocking down TrxR1 attenuated cell death in Tempol‑treated Wi‑38 VA‑13 cells but had mini‑ mal effects on A549 and Calu‑6  cells. Tempol treatment led to intracellular glutathione (GSH) depletion in various lung cell types. The degree of GSH depletion varied depending on Tempol concentration and cell type.

ROLE OF TPL IN CARDIO‑RESPIRATORY Systemic administration of TPl attenuates the cardio‑respiratory depressant effects of fentanyl According to Baby et  al., previous injection of TPl reduces the cardio‑­ respiratory effects of fentanyl without compromising its analgesic effects. As a result, TPl may not directly disrupt opioid‑receptors that elicit fentanyl effects. It is unclear if the effects of TPl are primarily attributable to changes in oxidative stress since the potent antioxidant, L‑NACme, had no impact on fentanyl‑induced reduction of respiration [33].

142  Chemical and Clinical Applications of Tempol

FIGURE 11.10  Role of TPL in heart disorders

Although the precise mechanism of action is uncertain, TPl may directly interfere (i.e., independent of superoxide/free radical scavenging) with intra‑ cellular signaling systems that cause fentanyl’s cardio‑respiratory depressive effects [31–33]. Figure 11.10 showed role of TPl in cardiac disorders. Figure  11.10 depicts the role of TPL in hear disorders. The oxidative stress responsible for myofilament protein oxidation, imbalance intracellular Ca2+ transport, Myofilament Ca2+ hypersensitivity, conversion of MIC alpha form to beta form, leads to cardiac dysfunctioning. TPl scavenge this stress and may be beneficial in prevention and treatment of diseses in progressive manner.

TPl relieves lung injury in a rat model of chronic intermittent hypoxia via suppression of inflammation and oxidative stress TPl treatment, according to Wang et al., relieved degenerative alterations in lung tissue, lowered leukocyte count, and protein content (P0.001) in bron‑ choalveolar lavage fluid (P0.001) (BALF) [34]. TPl inhibited the inflamma‑ tory response in lung tissue generated by IH, as demonstrated by lower levels of TNF‑, IL‑1, and IL‑6 (P0.001) and protein levels of COX‑2 and iNOS (P0.001). Furthermore, TPl reduced oxidative stress in lung tissue by decreas‑ ing MDA levels (P0.001) and increasing SOD activity (P0.001) and GSH

11  •  Tempol as reactive oxygen inhibitor  143 levels (P < 0.05). Furthermore, TPl suppressed the inflammatory response by inactivating the NF‑B pathway. Furthermore, the findings indicated that TPl inhibited oxidative stress by activating the Nrf2/HO‑1 pathway. According to the authors, TPl successfully cures OSA‑induced lung damage. Figures 15, 16 depicted the involvement of TPL in scavenging ROS and vasodilation by trapping nascent oxygen. TPl therapy reduced IH‑induced lung damage by decreasing the inflammatory response and oxidative stress. TPl’s protec‑ tive actions include the suppression of NF‑B and the activation of HO‑1/Nrf2 signaling pathways. This research might give evidence for TPl as a possible medicine for treating lung damage in OSAS patients [34]. Figure  11.11 demonstrates the impact of ROS imbalance on the liver, which leads to ACE damage and apoptosis, fibroblast recruitment, which leads to excessive cell accumulation, and production of inflammatory cells, which leads to prolonged inflammation, which leads to hypoxia. TPL protects the body by suppressing ROS, NF‑B, and stimulating the HO‑1/Nrf2 signal‑ ing pathways.

TPl reduces oxidative stress, improves insulin sensitivity, decreases renal dopamine D1 receptor hyperphosphorylation, and restores D1 receptor–G‑protein coupling and functioning obese Zucker rats According to Banday et al., oxidative stress promotes renal dopamine D1 recep‑ tor dysfunction in obese Zucker rats. TPl also increased receptor G‑protein coupling while decreasing D1 receptor phosphorylation. Dopamine inhibited Na‑K‑ATPase activity in TPl‑treated obese rats, but SKF‑38393 elicited a natri‑ uretic response. In obese Zucker rats, TPl decreases oxidative stress and enhances insulin sensitivity. As a consequence, D1 receptor hyperphosphorylation is reduced, restoring receptor–G‑protein coupling and the SKF‑38393 natriuretic response [35]. Figure 11.12 demonstrates the role of TPL in reducing oxidative stress, which causes mitochondrial damage through a number of mechanisms. Hepatocytes may be harmed by RONS produced in mitochondria. Excess O2 in mitochondria is used to create ATP through OXPHOS in the Electron Transport System, whereas a tiny quantity of O2• and nacent oxygen is pro‑ duced as a by‑product of the OXPHOS process under normal physiological circumstances. Active metabolites and other adverse stimuli may directly inter‑ fere with ETC, resulting in increased O2• production. NADH produced dur‑ ing the metabolic process is also carried into mitochondria, where it promotes

144  Chemical and Clinical Applications of Tempol

FIGURE 11.11  Effect of reactive oxygen species on lungs in redox homeostasis as well as imbalance

FIGURE 11.12  Role of TPL in reducing oxidative stress, enhancing insulin sensitivity, reducing dopamine and restoring D1 receptor‑Gprotein coupling

11  •  Tempol as reactive oxygen inhibitor  145 electron leakage. Excess free fatty acid interferes with the OXPHOS process by increasing the TCA cycle. Under the action of mitochondrial SOD, O2• cre‑ ates H2O2, which is then transformed to •OH radical via the Fenton reaction. Meanwhile, O2• may combine with NO from iNOS to generate ONOO. (G) Changes in membrane permeability (MPT) depending on membrane permea‑ bility pore (MPTP) and damage to mitochondrial DNA result in RONS release into the cytoplasm, which activates JNK1/2. Phosphorylated JNK is delivered to the mitochondria and disrupts ETC through Sab activity, resulting in the production of RONS. This damage cycle results in persistent activation of JNK and amplification of the oxidative stress impact [36].

ROLE OF TPL IN IMPROVING THE ROS IMBALANCE IN OBESITY Yamato et  al. (2016) studied the possible effect of TPl in reducing ROS imbalance in obese mice. Obesity is an adipose tissue condition that causes problems in energy metabolism. Redox mechanisms tightly manage constant energy transformation. NAD+, NADH, and NADPH are key components of the electron transport system in energy metabolism. Because TPL is a redox‑cycling nitroxide, it induces ROS scavenging and is thereafter converted to hydroxylamine through NADH. It is also involved in the ascorbic acid–glutathione redox pathway, where it generates NAD+. In light of the preceding TPl effect, Mayumi Yamato et al. described its function as anti‑ oxidants and NAD+/NADH modulators on metabolic imbalance in obese mice. When paired with a dietary intervention, transitioning from a high fat diet to a regular diet, increases in the NAD+/NADH ratio by TPL alleviated the metabolic imbalance. When compared to a control diet, plasma levels of the super oxide marker dihydroethidium were greater in mice following the dietary intervention, but were restored with TPL ingestion. These results provide new light on redox regulation in obesity [37].

CONCLUSION In conclusion, the potential therapeutic implications of ROS scavengers, particularly nitroxides like Tempol (TP), in mitigating oxidative stress and

146  Chemical and Clinical Applications of Tempol inflammation in various disease conditions. Studies have shown that Tempol exhibits antioxidant properties by scavenging free radicals, modulating redox signaling pathways, and protecting against oxidative damage in tissues and organs such as the retina, lungs, heart, and kidneys. Moreover, Tempol has been investigated for its potential therapeutic effects in conditions such as osteoarthritis, cerebral malaria, lung injury induced by chronic intermittent hypoxia, and even in mitigating the cardio‑respiratory depressant effects of opioids like fentanyl. Its ability to modulate oxidative stress, inflammation, and cellular redox dynamics makes Tempol a promising candidate for the treatment of various diseases associated with oxidative damage.

REFERENCES







1. Kurutas EB. The Importance of Antioxidants Which Play the Role in Cellular Response Against Oxidative/Nitrosative Stress: Current State. Nutr. J. 2016 Jul 25, 15 (1), 71. https://doi.org/10.1186/s12937‑016‑0186‑5 2. Schieber M, Chandel NS. ROS Function in Redox Signaling and Oxidative Stress. Curr. Biol. 2014 May 19, 24 (10), R453–R462. https://doi.org/10.1016/j. cub.2014.03.034 3. Cardoso MA, Gonçalves HMR, Davis F. Reactive Oxygen Species in Biological Media Are They Friend or Foe? Major In vivo and In vitro Sensing Challenges. Talanta 2023, 260, 124648. https://doi.org/10.1016/j.talanta.2023.124648 4. de Zambotti M, Trinder J, Silvani A, Colrain IM, Baker FC. Dynamic Coupling Between the Central and Autonomic Nervous Systems During Sleep: A Review. Neurosci. Biobehav. Rev. 2018, 90, 84–103. https://doi.org/10.1016/j. neubiorev.2018.03.027 5. Lettre G, Hengartner MO. Developmental Apoptosis in C. elegans: A Complex CEDnario. Nat. Rev. Mol. Cell Biol. 2006 Feb, 7 (2), 97–108. https://doi. org/10.1038/nrm1836. PMID: 16493416. 6. Eggler AL, Small E, Hannink M, Mesecar AD. Cul3‑mediated Nrf2 Ubiquitination and Antioxidant Response Element (ARE) Activation Are Dependent on the Partial Molar Volume at Position 151 of Keap1. Biochem. J. 2009 Jul 29, 422 (1), 171–180. https://doi.org/10.1042/BJ20090471 7. Raghunath A, Sundarraj K, Nagarajan R, Arfuso F, Bian J, Kumar AP, Sethi G, Perumal E. Antioxidant Response Elements: Discovery, Classes, Regulation and Potential Applications. Redox Biol. 2018 Jul, 17, 297–314. https://doi. org/10.1016/j.redox.2018.05.002 8. Tu W, Wang H, Li S, Liu Q, Sha H. The Anti‑Inflammatory and Anti‑Oxidant Mechanisms of the Keap1/Nrf2/ARE Signaling Pathway in Chronic Diseases. Aging Dis. 2019 Jun 1, 10 (3), 637–651. https://doi.org/10.14336/AD.2018.0513 9. Lewandowski M, Gwozdzinski K. Nitroxides as Antioxidants and Anticancer Drugs. Int. J. Mol. Sci. 2017 Nov 22, 18 (11), 2490. https://doi.org/10.3390/ ijms18112490

11  •  Tempol as reactive oxygen inhibitor  147 10. Mendonca M, Tarpey M, Krishna M, Mitchell JB, Welch WJ, Wilcox CS. Acute Antihypertensive Action of Nitroxides in the Spontaneously Hypertensive Rat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006, 290, R37–R43. 11. Wilcox CS. Effects of Tempol and Redox‑cycling Nitroxides in Models of Oxidative Stress. Pharmacol. Ther. 2010 May, 126 (2), 119–145. https://doi. org/10.1016/j.pharmthera.2010.01.003 12. Vorobjeva NV, Pinegin BV. Effects of the Antioxidants Trolox, Tiron and Tempol on Neutrophil Extracellular Trap Formation. Immunobiology 2016, 221, 208–219. https://doi.org/10.1016/j.imbio.2015.09.005 13. Zhao B, Pan Y, Wang Z, Tan Y, Song X. Intrathecal Administration of Tempol Reduces Chronic Constriction Injury‑Induced Neuropathic Pain in Rats by Increasing SOD Activity and Inhibiting NGF Expression. Cell. Mol. Neurobiol. 2016, 36, 893–906. https://doi.org/10.1007/s10571‑015‑0274–7. 14. Dickey JS, Gonzalez Y, Aryal B, Mog S, Nakamura AJ, Redon CE, Baxa U, Rosen E.], Cheng G, Zielonka J, et al. Mito‑tempol and Dexrazoxane Exhibit Cardioprotective and Chemotherapeutic Effects Through Specific Protein Oxidation and Autophagy in a Syngenic Breast Tumor Preclinical Model. PLoS One 2013, 8, e70575. https://doi.org/10.1371/journal.pone.0070575 15. Gwozdzinski K., Bartosz G. Nitroxide Reduction in Human Red Blood Cells. Curr. Top. Biophys. 1996, 20, 60–65. 16. Gwozdzinski K, Bartosz G, Leyko W. Effect of Gamma Radiation on the Transport of Spin‑labeled Compounds Across the Erythrocyte Membrane. Radiat. Environ. Biophys. 1981, 19, 275–285. https://doi.org/10.1007/BF01324093 17. Gadjeva V, Kuchukova D, Tolekova A, Tanchev S. Beneficial Effects of Spin‑labelled Nitrosourea on CCNU‑induced Oxidative Stress in Rat Blood Compared with Vitamin E. Pharmazie 2005, 60, 530–532. 18. Nilsson UA, Olsson LI, Carlin G, Bylund‑Fellenius AC. Inhibition of Lipid Peroxidation by Spin Labels: Relationships Between Structure and Function. J. Biol. Chem. 1989, 264, 11131–11135. 19. Glebska J, Gwozdzinski K. Oxygen‑dependent Reduction of Nitroxides by Ascorbic Acid and Glutathione. EPR Investigations. Curr. Top. Biophys. 1998, 22, 75–82. 20. Brennan ML, Hazen SL Amino Acid and Protein B Oxidation in Cardiovascular Disease. Amino Acids 2003, 25, 365374. https://doi.org/10.1007/ s00726‑003‑0023‑y 21. Kris‑Etherton PM, Lichtenstein AH, Howard BV, Steinberg D, Witztum JL. Antioxidant Vitamin Supplements and Cardiovascular Disease. Circulation 2004, 110, 637641. https://doi.org/10.1161/01.CIR.0000137822.39831.F1 22. Szabo C, Ischiropoulos H, Radi R. Peroxynitrite: Biochemistry, Pathophysiology and Development of Therapeutics. Nat. Rev. Drug Discovery 2007, 6, 662680. 23. Augusto O, Bonini MG, Amanso AM, Linares E, Santos CXC, De Menezes SL. Nitrogen Dioxide and Carbonate Radical Anion: Two Emerging Radicals in Biology. Free Radic. Biol. Med.  2002, 32, 841859. https://doi.org/10.1016/ s0891–5849(02)00786‑4 24. Wipf P, Xiao J, Jiang J, Belikova NA, Tyurin VA, Fink MP, Kagan VE. Mitochondrial Targeting of Selective Electron Scavengers: Synthesis and Biological Analysis of Hemigramicidin‑TPL Conjugates. J. Am. Chem. Soc. 2005, 127, 12460–12461. https://doi.org/10.1021/ja053679l

148  Chemical and Clinical Applications of Tempol 25. Halliwell B. Biochemistry of Oxidative Stress. Biochem. Soc. Trans. 2007 Nov, 35 (Pt 5), 1147–1150. https://doi.org/10.1042/BST0351147 26. Sies H. Oxidative Stress: A Concept in Redox Biology and Medicine. Redox Biol. 2015, 4, 180–183. https://doi.org/10.1016/j.redox.2015.01.002. 27. Stadtman ER, Levine RL. Free Radical‑mediated Oxidation of Free Amino Acids and Amino Acid Residues in Proteins. Amino Acids. 2003 Dec, 25 (3–4), 207–218. https://doi.org/10.1007/s00726‑003‑0011–2 28. Tanito M, Li F Anderson RE. Protection of Retinal Pigment Epithelium by OT‑551 and Its Metabolite TEMPOL‑H Against Light‑Induced Damage in Rats. Exp. Eye Res. 2010, 91 (1), 111–114. https://doi.org/10.1016/j.exer.2010.04.012 29. Balaraman K. Reactive Oxygen Species, Pro‑inflammatory and Immunosuppressive Mediators Induced in COVID‑19: Overlapping Biology with Cancer. RSC Chem. Biol. 2021, 2, 1402. https://doi.org/10.1039/d1cb00042j 30. Calabrese G, Ardizzone A, Campolo M, Conoci S, Esposito E, Paterniti I. Beneficial Effect of TPl, a Membrane‑Permeable Radical Scavenger, on Inflammation and Osteoarthritis in In Vitro Models. Biomolecules 2021, 11 (3), 352. https://doi.org/10.3390/biom11030352. 31. Banday AA, Marwaha A, Lakshmi S, Tallam F, Mustafa FL. TPl Reduces Oxidative Stress, Improves Insulin Sensitivity, Decreases Renal Dopamine D1 Receptor Hyperphosphorylation, and Restores D1Receptor–G‑Protein Coupling and Functionin Obese Zucker Rats. Diabetes 2005, 54, 2219–2226. https://doi.org/10.2337/diabetes.54.7.2219. 32. Park WH. Tempol Differently Affects Cellular Redox Changes and Antioxidant Enzymes in Various Lung‑related Cells. Sci. Rep. 2021 Jul 21, 11 (1), 14869. https://doi.org/10.1038/s41598‑021‑94340‑z 33. Baby S, Gruber R, Discala J, Puskovic V, Jose N, Cheng F, Jenkins M, Seckler J, Lewis S. Systemic Administration of TPl Attenuates the Cardiorespiratory Depressant Effectsof Fentanyl. Front. Pharmacol. 2021, 12, 690407. https:// doi.org/10.3389/fphar.2021.690407 34. Wang Y, Hai B, Ai L, Cao Y, Li R, Li H, Li Y. TPl Relieves Lung Injury in a Rat Model of Chronic Intermittent Hypoxia via Suppression of Inflammation and Oxidative Stress. Iran. J. Basic Med. Sci. 2018, 21, 1238–1244. https://doi. org/10.22038/ijbms.2018.31716.7714 35. Banday AA, Lau YS, Lokhandwala MF. Oxidative Stress Causes Renal Dopamine D1 Receptor Dysfunction and Salt‑sensitive Hypertension in Sprague‑Dawley Rats. Hypertension 2008 Feb, 51 (2), 367–375. https://doi. org/10.1161/HYPERTENSIONAHA.107.102111 36. Li F, Jiang C, Krausz K, Li Y, Albert I, Hao H. Microbiomeremodelling Leads to Inhibition of Intestinal Farnesoid X Receptor Signalling and Decreased Obesity. Nat. Commun. 2013, 4, 2384. https://doi.org/10.1038/ncomms3384 37. Yokota TM, Kinugawa S, Hirabayashi K, et  al. Systemic Oxidative Stress is Associated with Lower Aerobic Capacity and Impaired Skeletal Muscle Energy Metabolism in Heart Failure Patients. Sci. Rep. 2021, 11, 2272. https:// doi.org/10.1038/s41598‑021‑81736‑0

Nano‑ formulations of Tempol

12

Abhishek Tiwari1*, Varsha Tiwari2*, and Bimal Krishna Banik3* INTRODUCTION Nanotechnology holds immense importance in the medical realm, par‑ ticularly concerning lipid‑insoluble drugs. Various nano‑formulations have evolved over the years to address this need. These formulations include Polymeric particles (1989), PEGylation (1990), lipid disks (1995), Nano/ Microemulsions (1995), Iron oxide nanoparticles (1996), Hydrogels (2002), Polymeric micelles (2003), Nano‑crystals (2003), albumin nanoparticles (2005), Cell‑based therapies (2010), viral nanoparticles (2010), Oncolytic viruses (2015), and Lipid‑based nucleic acid nanoparticles (2018). These advancements have significantly impacted the treatment of various disorders, particularly in delivering both water and lipid‑­soluble drugs and phytocon‑ stituents. Figure 12.1 shows various nano‑formulations Tempol in the treat‑ ment of Liver injury, inflammation, and ROS. Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 2 Department of Pharmacognosy, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 3 Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University, Al Khobar 31952, Kingdom of Saudi Arabia; 1

*

Corresponding Authors: [email protected]; [email protected]; [email protected]

DOI: 10.1201/9781003426820-12149

150  Chemical and Clinical Applications of Tempol

FIGURE 12.1  Development of nano‑formulations since 1989–2018

UTILIZING TP‑OXIDIZED NANO‑CELLULOSE FOR SILVER NANOPARTICLE FORMULATIONS Pawcenis et al. 2022, revealed the effectiveness of TOCNs and its fractions in designing of AgNPs. Aqueous suspension of AgNO3 containing TOCN was heated to design AgNPs. The researchers developed three distinct nanocom‑ posite materials, each utilizing a different TOCN fraction, which were then reduced in situ by TOCN‑synthesized AgNPs. Water‑soluble fractions give rise to AgNPs, whereas insoluble fractions leads to NPs with non‑uniform size [1].

12  •  Nano‑formulations of Tempol   151

MICRO‑ AND NANO‑FORMULATIONS OF CMC, DIALDEHYDE, AND TP‑OXIDIZED CELLULOSE (TPOC) FOR ANTIMICROBIAL AND WOUND TREATMENT Alavi et  al., discussed the challenges associated with treating persistent infected wounds namely DFU, in immune compromised patients. As the emergence of ABR bacteria and fungi, traditional medications may be inef‑ fective in future microbial infections at numerous sites. Therefore, biomateri‑ als, CMC, dialdehyde and TPOC may show promising effect in combating this challenge against microorganisms [2].

TPOBC WITH AGNPS FOR WOUND HEALING Wu et al. explored the potential of utilizing bio‑compatible BCP in wound dress‑ ings. Despite its biocompatibility, it lacks inherent antibacterial activity, required for further modification for biomedical applications. They reported the develop‑ ment of TOBCP with C6‑RCOO‑ groups through TP‑mediated oxidation leads to the development of AgNP and nanofibers. These showed excellent biocom‑ patibility and antibacterial potential for Escherichia  coli and Staphylococcus aureus, may be an potential choice of drug against microorganisms [3].

SURFACE AMENDMENT OF TP Elżbieta et  al. revealed surface modification techniques employing stable radicals, specifically TP and fractions. This chapter delineates two pri‑ mary approaches. Firstly, they immobilize TP groups onto surface of silicon wafers, nanomaterials, organic polymers, etc. Secondly, they explored the utilization of nitroxide mediated radical polymerization (NMRP) to graft multiple chains of polymer, designs polymer brushes on nanostructured sur‑ faces. They also investigate the effect of polymer modifications on numerous physicochemical parameters [4].

152  Chemical and Clinical Applications of Tempol FIGURE 12.2  A, The immobilization of TP on silica surface TP trapping through sol‑gel technique; B, The SBA‑15 silica linked with TP; C, The designing of silica improved with TP through silane‑coupling procedure; D, Organically linked TP through sol‑gel method

12  •  Nano‑formulations of Tempol   153

Silica associated Tempol Tsubokawa et al. [5], very first reported the TP immobilization on silica par‑ ticles, treated with TSPA converts surface OH group into RC(=O)OC(=O)R group. The immobilization of TP radicals was achieved through the reaction of TP with surface groups in the presence of N,N′‑dicyclohexyl carbodiimide (DCCI) (Figure 12.2). Brunel et al. [6] designed mesoporous silica to immobilize TP. This silica material is possess hexagonal network with particle size of 15–100 Å. These silica supports immobilized TP due to superior pore size, internal surface and silanol groups [7]. Brinker describes the Stöber method in the transformation of colloidal solutions from precursors lead to gelation removal of liquids results in porous monolithic substances using silica based tempol derivatives (3) [8]. This method includes the use of liquid alkoxysilanes like Si(OR)4 as precursors that can be readily hydrolyzed into Si(OH)4, which on polycondensation, results in three‑dimensional network. Water and alcohol remain entrapped within the network removed subsequently. Through drying and aging processes [9]. Ciriminna et  al., very first reported the silica modified TP synthesis through sol‑gel technique, which has been further proved efficient as catalyst in oxidation of sugars into urinates (4) [10]. They utilized a TP precursor through amination between oxo‑TP and trimethoxy silane. Afterward, TP‑derived alk‑ oxysilane was merged into silica surface through sol‑gel technique using H+/ OH− followed by co‑polycondensation with Si(OCH3)4 as depicted in (5) [11].

Adsorbed TP on metal and its oxide NPs These MNP’S imparts numerous benefits, i.e., stability, functionality, solubil‑ ity, self‑assemble, etc. Moreover, these also show promising potential support for functional foods especially catalyst, in view of large surface area and synergistic effects [12]. Karimi et  al. [13] revealed an EASA method [14] to design coating of mesoporous silica MCM‑41 onto electrodes functionalized with TP. Firstly, thin film of APS has been deposited on the surface of graphite electrode by condensation among functionalized/non‑functionalized precursors triethoxysilane and TEOS in the presence of CTAB as structure‑­modifying agent. Figure 12.2 depicts the reductive amination of APS in the presence of NaBH3CN in presence of silica‑substituted electrodes (Figure 12.3). Swiech et  al., designed N‑AuNPs on gold electrode surface using a 1,9‑nonanedithiol linker. This electrode has been used in electrocatalytic oxidation of C6H5CH2OH to aldehyde. The catalytic effectiveness of the

154  Chemical and Clinical Applications of Tempol

FIGURE  12.3  A, The pathway of electrode modification with a thin layer of TP‑functionalized ordered mesoporous silica (TGSE); B, The diagram depicts an electrode modified with N‑AuNPs acquired using as a stabilizing agent, and an electrode modified with a monolayer obtained via the direct chemisorption of on a flat gold surface

N‑AuNPs has been compared with that of nitroxides monolayer formed through direct chemisorption on Au surface (Figure 12.3) [15].

Carbon nanomaterials grafted with TEMPO Zhao et al. [16], first reported the surface modification of carbon nanotubes (CNTs) with TP derivatives. They oxidized MWNT by treating with mix‑ ture of HNO3/H2SO4. These COOH group were further transformed into COCl2 taking SOCl2. This reaction has been applied to TP as depicted in Figure 12.4, Figure 8 [16, 17].

12  •  Nano‑formulations of Tempol   155

FIGURE 12.4  A, The scheme of multi‑walled carbon nanotubes (MWNTs) modification with TP; B, Route to synthesis of TP‑functionalized MWNTs

FIGURE 12.5  The procedure for the synthesis of graphene grafted with TP

Yang et  al. [18], designed 4‑step technique for developing TP‑­ functionalized MWNTs. The researchers includes sequential stages namely carbon nanotubes subjected to oxidation taking. Their procedure involved several sequential stages: initially, the carbon nanotubes underwent oxida‑ tion using C5H6O4 followed by subsequent COOH attachment on the surface of MWNTs via coupling with C3H7BrO in the presence of DCC. The bromo atom transferred to azo group. This leads to azide/alkyne copper(I) formation through “click” reaction as depicted in Figure 12.4, Figure 9.

Graphene (G) and graphene oxide (GO) Bosch‑Navarro et al. [19], designed graphene‑linked TP through Bingel‑Hirsch reaction taking graphene as starting material. Graphene was designed by directly shedding graphite in oDCB/C6H5CH2NH2 with the aid of ultrasonic radiation (Figure 12.5).

156  Chemical and Clinical Applications of Tempol

SILVER NPS SYNTHESIS ON TP‑SUBSTITUTED BACTERIAL CELLULOSE Elayaraja et  al. [20] investigate the TP oxidation to activate COOH group, lead to BC oxidation in AgNO3 results in AgNP with BC in order to trig‑ ger vibriocidal potential. It further leads to evaluation through SEM, EDS, and XRD. They have also assessed the vibriocidal potential against V. para‑ haemolyticus and V. harveyi revealed AgNP revealed greater effectiveness against above pathogens, therefore may be the promising therapy in manage‑ ment of shrimp pathogens [4].

TNFC FOR CONTROLLED RELEASE AGAINST ANTIMICROBIAL CU Cu NPs were designed from using CuSO4 (0%, 30%, 50%, and 70%) with respect to cellulose‑Cu material mass. This mixture was then treated with

FIGURE  12.6  Different formulations of TP include silica particles, magnetic nanoparticles, metallic nanoparticles, carbon nanotubes, graphene, polymer brushes, nanohybrids, fullerenes, inorganic flat surfaces, and polymers

12  •  Nano‑formulations of Tempol   157 PVA to form thin film, this film was subjected to antibacterial potential of against E. coli. They revealed that by enhancing the cellulose content Cu amount can be regulated, it depicts that it follows the power law as when the concentration is 30% or low exponential release of Cu has been observed whereas when it has been raised to 70%, the conc. of Cu has been decreased. Figure 12.6 depicts the various compositions of the cellulose‑Cu hybrid mate‑ rial [21].

BRAIN‑TARGETED DELIVERY OF TP‑LOADED NANOPARTICLES FOR NEUROLOGICAL DISORDERS Nanoprecipitation of TP on PLGA to target brain disorders, namely Parkinson’s and Alzheimer’s have been designed. These conditions may be specified an increase in ROS and these TP NPs may act as protective effect in manage‑ ment of the above disorders. They have loaded NPs with TP and transfer‑ rin antibodies, covalently with PLGA NPs using NHS‑PEG3500‑Maleimide crosslinker in order to make the sustained release formulation. Anticancer potential of these NPs have been evaluated through MTT assays which further lessen cell death in RG2  cells and compared with that of conjugated and unconjugated TPL. The results revealed that TP‑conjugated NPs showed potential for the management of neurodegen‑ erative disorders [22].

NES FOR HAIR LOSS A novel NEG targeting mechanisms of inflammation and apoptosis. It includes the formulation development of NE’s for hair loss. The NEG has been for‑ mulated for topical use by taking TP and cyclosporin‑A. Pharmacological analysis revealed that this formulation delivered the drug efficiently, inside the dermis. In‑vivo study was further performed which promotes the hair growth, color intensity further confirmed by histological analysis. On the basis of this study, CsA‑Tempol was found to possess significant therapeutic platform for the management of alopecia [23]. This is another novel use of tempol‑derived NES in maintain the hair growth.

158  Chemical and Clinical Applications of Tempol

FIGURE 12.7  Tempol‑loaded nanoparticles in treatment of epidermoid cancer

NF’S AGAINST MDRC Multidrug resistance is becoming an alarming factor in chemotherapy over 90% of patients. This resistance is associated with reactive oxygen species (ROS)‑regulated drug efflux proteins, specifically P‑glycoprotein (P‑gp) and multidrug resistance‑associated protein 1 (MRP1). The multidrug resistance by utilizing nanoparticles containing ROS‑scavenging nitroxide radicals, termed RNPN (pH‑sensitive) and RNPO (pH‑insensitive), in combination with the conventional chemotherapy drug doxorubicin (Dox), in drug‑­resistant epidermoid cancer cell lines KB‑C2 (P‑gp expressing) and KB/MRP (MRP1 expressing) have been reported. Moreover, RNP treatment effectively disrupted crucial ROS signaling pathways, downregulates ROS‑regulated drug efflux protein expression (P‑gp and MRP1), thus sensitizing resistant cells to Dox. These results underscore the potential of ROS‑scavenging RNPs as promising therapeutic candidates for overcoming drug resistance in multidrug‑resistant cancers. Figure  12.7 depicts the design of TP‑loaded NPs in cancer, illustrating the suppression of resistance and the promotion of sensitivity [24].

NANOPLATFORM TARGETING NUMEROUS ROS WITHIN THE BRAIN Zhang et  al., developed a nanomedicine possessing properties to SOD and catalase by merging H2O2‑neutralizing substance, Oxb CD, with TP, referred

12  •  Nano‑formulations of Tempol   159

FIGURE 12.8  Development of SOD/catalase mimetic nanomedicine; Demonstrates two described methods for surface modification employing TEMPO and its derivatives: A, utilizing readily available nitroxyl groups, and B, incorporating nitroxyl groups connected with polymer chains. The linker molecule, denoted as Y, facilitates direct attachment to the surface.

160  Chemical and Clinical Applications of Tempol as TP/OxbCD NP. It exhibits potential therapy against both acute/chronic colitis. Furthermore, in addition to act as functional, efficient, and safe nanocarrier for TP, OxbCD NP offers promising mode for ROS‑‑responsive transfer of therapeutic agents, namely peptides, proteins, and nucleic acids for inflammatory bowel disease (IBD) and associated intestinal diseases. Figure  13 depicts the developmental stages of the mimetic nanomedicine containing SOD/catalase [25] (Figure 12.8).

Immobilization of TP onto inorganic surfaces Over the past 20 years, there has been significant research focus on attaching TP molecules to various inorganic substrates like silica, metals, and metal oxides, especially for catalytic purposes. TP and its derivatives have shown effectiveness as catalysts for oxidizing alcohols, diols, and sugars in homoge‑ neous systems. Typically, this process involves a small amount of TP (around 1  mol%) along with a stoichiometric quantity of a terminal oxidant (co‑­ oxidant). The co‑oxidant plays a crucial role in regenerating oxoammonium ions from hydroxylamine. Various systems function as terminal oxidants for this purpose, including hypochlorite with bromides (Anelli protocol), bis(acetoxyiodo)benzene (Margarita protocol), meta‑chloroperbenzoic acid (m‑CPBA), N‑chlorosuccinimide, oxone, and oxygen with CuCl or ruthe‑ nium complexes. Importantly, electrochemical methods can also regenerate oxoammonium ions from nitroxide, offering an environmentally friendly approach (14). Immobilizing the catalyst and/or terminal oxidant onto sur‑ faces facilitates straightforward separation and recycling, making it highly advantageous from both economic and environmental standpoints [5].

CONCLUSION The utilization of TP and its derivatives for surface modification has emerged as a promising avenue in various scientific disciplines. The two discussed approaches, involving either easily accessible nitroxyl groups or nitroxyl groups connected with polymer chains, offer versatile methods for enhanc‑ ing surface properties and functionality. The linker molecule, denoted as Y, serves as a crucial component in facilitating direct connection with the sur‑ face, thereby enabling precise control over surface modification processes. The catalytic potential of TP and its derivatives has been demonstrated in cataly‑ sis, drug delivery, and nanotechnology, among other fields. By leveraging their ROS‑scavenging capabilities, these compounds hold significant promise for

12  •  Nano‑formulations of Tempol   161 mitigating oxidative stress‑related damage and advancing therapeutic inter‑ ventions for various diseases. Further research and development efforts are warranted to fully exploit the potential of TP‑based surface modification techniques. This includes exploring novel applications, optimizing synthesis methods, and investigating the scalability and practicality of these approaches. With continued innovation and collaboration across multidisciplinary fields, TP‑based surface modification strategies are poised to make significant contri‑ butions to advancements in surface engineering and functional material design.

REFERENCES 1. Pawcenis D, Twardowska E, Leśniak M, Jędrzejczyk RJ, Sitarz M, Profic‑Paczkowska J. TEMPO‑oxidized Cellulose for In situ Synthesis of Pt Nanoparticles. Study of Catalytic and Antimicrobial Properties. Int. J. Biol. Macromol. 2022, 213, 738–750. https://doi.org/10.1016/j.ijbiomac.2022.06.020 2. Alavi M, Asare‑Addo K, Nokhodchi A. Lectin Protein as a Promising Component to Functionalize Micelles, Liposomes and Lipid NPs against Coronavirus. Biomedicines 2020, 8, 580. https://doi.org/10.3390/biomedicines8120580 3. Wu CN, Fuh S‑C, Lin S‑P, Lin Y‑Y, Chen H‑Y, Liu J‑M. TEMPO‑Oxidized Bacterial Cellulose Pellicle with Silver Nanoparticles for Wound Dressing. Biomacromolecules 2018, 19, 2, 544–554. 4. Elżbieta M. Surface Modification Using TEMPO and Its Derivatives. Adv. Colloid Interface Sci. 2017, 250, 158–184. https://doi.org/10.1016/j.cis.2017. 08.008 5. Tsubokawa N, Kimoto T, Endo T. Oxidation of Alcohols with Copper (II) Salts Mediated by Nitroxyl Radicals Immobilized on Ultrafine Silica and Ferrite Surface. J. Mol. Catal. A Chem. 1995, 101, 45–50. 6. Brunel D, Fajula F, Nagy J, Deroide B, Verhoef M, Veum L, et al. Comparison of Two MCM‑41 Grafted TEMPO Catalysts in Selective Alcohol Oxidation. Appl. Catal. A 2001, 213, 73–82. 7. Wight A, Davis M. Design and Preparation of Organic‑Inorganic Hybrid Catalysts. Chem. Rev. 2002, 102, 3589–3614. 8. Brinker CJ. GW Scherer Sol‑Gel Science. Academic: San Diego, 1990. 9. Hench LL, West JK. The Sol‑Gel Process. Chem. Rev. 1990, 90, 33–72. 10. Ciriminna R, Pagliaro M, Blum J, Avnir D. Sol–Gel Entrapped TEMPO for the Selective Oxidation of Methyl α‑D‑Glucopyranoside. Chem. Commun. 2000, 1441–1442. 11. Tanaka H, Kuroboshi M, Goto K. Electrooxidation of Alcohols in N‑Oxyl‑ Immobilized Silica Gel/Water­disperse System: Approach to Totally Closed System. Synthesis 2009, 2009, 903–908. 12. Fedlheim DL, Foss CA. Metal Nanoparticles: Synthesis, Characterization, and Applications. CRC Press: Boca Raton, 2001. https://www.taylorfrancis.com/ books/mono/10.1201/9780367800475/metal‑nanoparticles‑daniel‑fedlheim‑ colby‑foss.

162 Chemical and Clinical Applications of Tempol 13. Karimi B, Biglari A, Clark JH, Budarin V. Green, Transition‑Metal‑Free Aerobic Oxidation of Alcohols Using a Highly Durable Supported Organocatalyst. Angew. Chem. Int. Ed. Engl. 2007, 46, 7210–7213. 14. Walcarius A, Sibottier E, Etienne M, Ghanbaja J. Electrochemically Assisted Selfassembly of Mesoporous Silica Thin Films. Nat. Mater. 2007, 6, 602–608. 15. Swiech O, Bilewicz R, Megiel E. TEMPO Coated Au Nanoparticles: Synthesis and Tethering to Gold Surfaces. RSC Adv. 2013, 3, 5979–5986. 16. Zhao X, Lin W, Song N, Chen X, Fan X, Zhou Q. Water Soluble Multi‑walled Carbon Nanotubes Prepared via Nitroxide‑mediated Radical Polymerization. J. Mater. Chem. 2006, 16, 4619. 17. Zhao X‑D, Fan X‑H, Chen X‑F, Chai C‑P, Zhou Q‑F. Surface Modification of Multiwalled Carbon Nanotubes via Nitroxide‑mediated Radical Polymerization. J. Polym. Sci. A Polym. Chem. 2006, 44, 4656–4667. 18. Yang C, Guenzi M, Cicogna F, Gambarotti C, Filippone G, Pinzino C, et al. Grafting of Polymer Chains on the Surface of Carbon Nanotubes via Nitroxide Radical Coupling Reaction. Polym. Int. 2016, 65, 48–56. 19. Bosch‑Navarro C, Busolo F, Coronado E, Duan Y, Martí‑Gastaldo C, Prima‑Garcia H. Influence of the Covalent Grafting of Organic Radicals to Graphene on Its Magnetoresistance. J. Mater. Chem. C 2013, 1, 4590. 20. Elayaraja K, Zagorsek F, Xiang LJ. In Situ Synthesis of Silver Nanoparticles into TEMPO‑mediated Oxidized Bacterial Cellulose and their Antivibriocidal Activity Against Shrimp Pathogens. Carbohydrate Polymers,2017, 166, 329– 337. https://doi.org/10.1016/j.carbpol.2017.02.093 21. Jiang C, Oporto GS, Zhong T, et al. TEMPO Nanofibrillated Cellulose as Template for Controlled Release of Antimicrobial Copper from PVA Films. Cellulose 2016, 23, 713–722. https://doi.org/10.1007/s10570‑015‑0834‑5 22. Pinheiro RGR, Coutinho AJ, Pinheiro M, Neves AR. Nanoparticles for Targeted Brain Drug Delivery: What Do We Know? Int. J. Mol. Sci. 2021 Oct 28, 22 (21), 11654. https://doi.org/10.3390/ijms222111654 23. Deng Y, Huang F, Wang J, Zhang Y, Zhang Y, Su G, Zhao Y. Hair Growth Promoting Activity of Cedrol Nanoemulsion in C57BL/6 Mice and Its Bioavailability. Molecules 2021 Mar 23, 26 (6), 1795. https://doi.org/10.3390/ molecules26061795. 24. Emran TB, Shahriar A, Mahmud AR, Rahman T, Abir MH, Siddiquee MF. Multidrug Resistance in Cancer: Understanding Molecular Mechanisms, Immunoprevention and Therapeutic Approaches. Front. Oncol. 2022 Jun 23, 12, 891652. https://doi.org/10.3389/fonc.2022.891652 25. Zhang Q, Tao H, Lin Y, Hu Y, An H, Zhang D, Feng S, Hu H, Wang R, Li X, Zhang J. A Superoxide Dismutase/Catalase Mimetic Nanomedicine for Targeted Therapy of Inflammatory Bowel Disease. Biomaterials. 2016 Oct, 105, 206–221. https://doi.org/10.1016/j.biomaterials.2016.08.010

13

Safe handling, storage, and disposal of 4‑Hydroxy‑ TEMPO in compliance with pharmaceutical regulations

Abhishek Tiwari1*, Varsha Tiwari2*, and Bimal Krishna Banik3*

Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 2 Department of Pharmacognosy, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida-201313, Uttar Pradesh (India) 3 Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University, Al Khobar 31952, Kingdom of Saudi Arabia; 1

*

Corresponding Authors: [email protected]; [email protected]; [email protected]

DOI: 10.1201/9781003426820-13163

164  Chemical and Clinical Applications of Tempol

INTRODUCTION 4‑Hydroxy‑TEMPO is a stable nitroxide free radical compound utilized in various biochemical and physiological studies due to its radical scavenging and nitric oxide spin trapping properties. Its versatility extends to inducing oxidative stress, enhancing specific cellular proteins, reducing organ injury, exhibiting radio‑protective effects, and suppressing proliferation. When handling 4‑Hydroxy‑TEMPO, it’s crucial to adhere to pharmaceutical regu‑ lations to ensure safety, efficacy, and compliance. 4‑Hydroxy‑TEMPO is a stable, cell‑permeable nitroxide free radical. It acts as a radical scaven‑ ger and nitric oxide spin trap. Exhibits dose‑dependent effects on oxidative stress and cellular protein levels. Reported benefits include reducing organ injury and demonstrating radio‑protective and anti‑proliferative proper‑ ties. Wear appropriate personal protective equipment (PPE) such as gloves, lab coat, and safety goggles when handling 4‑Hydroxy‑TEMPO. Work in a well‑ventilated area to minimize inhalation risks. Avoid contact with skin, eyes, and mucous membranes. Clean spills promptly and dispose of waste properly [1–3].

PHYSICAL PROPERTIES 4‑Hydroxy‑TEMPO, commonly known as tempol, presents distinctive physi‑ cal properties that are integral to its applications and handling. When crystal‑ lized, tempol forms dark orange crystals, a characteristic likely attributed to the compound’s molecular structure, which absorbs specific wavelengths of light, resulting in the observed coloration. These crystals signify a high level of purity, crucial for ensuring the compound’s efficacy in various applica‑ tions. With a melting point ranging between 69°C and 71°C, tempol transi‑ tions from a solid to a liquid state within this temperature range. Consistency in its melting point aids in verifying its purity and facilitates its controlled transformation into different forms for specific uses. Its flash point, measured at 146°C, highlights the temperature at which tempol may emit flammable vapors, necessitating cautious handling and storage procedures to mitigate fire hazards. Notably, tempol demonstrates exceptional water solubility, with a solubility of 629.3 g/L at 20°C, rendering it completely soluble in water. This high solubility is advantageous for formulations requiring aqueous solutions

13  •  Safe handling and disposal of 4-HT   165 and facilitates its applications in various fields, such as pharmaceuticals and biochemistry. Furthermore, the density of tempol, recorded at 1.127 g/cm³ at 20°C, denotes its compactness and provides insight into its concentration in solutions. These physical properties collectively underscore tempol’s versatil‑ ity and suitability for diverse scientific and industrial endeavors, while also guiding its safe handling and storage practices [1–3].

STORAGE To properly store 4‑Hydroxy‑TEMPO (tempol), meticulous attention to storage conditions is imperative. Begin by selecting a tightly sealed con‑ tainer crafted from a material compatible with the compound, such as glass or high‑quality plastic. Ensure the container is securely sealed to prevent air or moisture ingress. Guard against light exposure by stor‑ ing the container away from direct sunlight or UV sources, as light can catalyze degradation. Similarly, shield the compound from heat sources, such as radiators or sunlight, as elevated temperatures hasten degradation processes. Maintain a dry environment to prevent moisture‑induced deg‑ radation or clumping. Moreover, ascertain that the storage area is devoid of incompatible materials that may react with 4‑Hydroxy‑TEMPO. To uphold stability and extend shelf life, regulate the storage temperature rigorously at −20°C (−4°F) using a freezer or refrigeration unit. Regular monitoring of storage conditions, including temperature fluctuations and signs of contamination or degradation, is indispensable for preserving the compound’s integrity. Adhering to these meticulous storage guide‑ lines will ensure the efficacy and longevity of 4‑Hydroxy‑TEMPO for its intended applications [1–3].

DISPOSAL Disposing of 4‑Hydroxy‑TEMPO (tempol) and its solutions requires meticu‑ lous adherence to local regulations and guidelines to prevent environmental harm and ensure compliance. Begin by ascertaining whether the substance and its solutions qualify as hazardous waste according to local laws, con‑ sidering their potential impact on human health and the environment.

166  Chemical and Clinical Applications of Tempol Once identified as hazardous waste, employ appropriate containers specifi‑ cally designed for hazardous materials and clearly label them to indicate their contents and associated hazards, including the name of the substance (4‑Hydroxy‑TEMPO). Next, initiate contact with authorized waste manage‑ ment facilities or hazardous waste disposal services in your area to arrange for proper disposal. These facilities possess the expertise and equipment necessary to handle hazardous waste safely and in accordance with regula‑ tions. Ensure secure sealing of containers during transportation to prevent spills or leaks. Keep detailed records of the disposal process for regulatory ­compliance purposes. Never dispose of 4‑Hydroxy‑TEMPO or its solutions improperly by discarding them in regular trash or pouring them down the drain, as this can lead to environmental contamination and regulatory viola‑ tions. By meticulously following these steps and adhering to local regula‑ tions, you can ensure the safe and responsible disposal of 4‑Hydroxy‑TEMPO and its ­solutions [1–3].

SAFETY MEASURES Hazards 4‑Hydroxy‑TEMPO (tempol) presents several significant hazards, as classi‑ fied by hazard statements and toxicity categories. Firstly, it poses an acute toxicity risk if ingested orally, categorized as Category 4, according to hazard statement H302. This indicates that while harmful effects are possible upon ingestion, they are generally less severe compared to substances categorized in higher toxicity categories. Secondly, tempol is classified under Category 1 for serious eye damage, denoted by hazard statement H318. This signifies that direct contact with the compound or its solutions can result in severe eye irritation or corrosion, requiring immediate medical attention to prevent last‑ ing damage. Additionally, tempol presents a hazard of specific target organ toxicity from repeated exposure via oral ingestion, categorized as Category 2. Hazard statement H373 highlights the potential for prolonged or repeated exposure to cause damage to specific organs, particularly the liver and spleen. This emphasizes the importance of minimizing exposure and implementing stringent safety measures to mitigate health risks associated with handling tempol. Overall, these hazards underscore the necessity of handling tempol with utmost care, adhering to safety protocols, and ensuring proper storage, handling, and disposal procedures to safeguard both human health and the environment [1–3].

13  •  Safe handling and disposal of 4-HT   167

PRECAUTIONS 4‑Hydroxy‑TEMPO (tempol) poses various hazards that necessitate careful handling and adherence to safety precautions outlined by hazard statements. Firstly, exposure to tempol dust should be avoided to prevent inhalation, as indicated by hazard statement P260, which advises against breathing dust par‑ ticles. Upon contact with skin, thorough washing is essential to remove any residue and prevent potential irritation or adverse reactions, as emphasized by hazard statement P264. Additionally, wearing appropriate eye protection or face protection, as stated in hazard statement P280, is crucial to safeguard against eye exposure and minimize the risk of serious eye damage. In the event of ingestion and subsequent feelings of illness, immediate action is advised, including contacting a poison center or seeking medical assistance, per hazard statement P301 + P312. Eye exposure to tempol requires prompt rinsing with water for several minutes, removal of contact lenses (if applicable and easily done), and continued rinsing, as instructed by hazard statement P305 + P351 + P338, to mitigate the risk of eye damage. Lastly, if any adverse symptoms persist or worsen, seeking medical advice and attention is paramount, as out‑ lined by hazard statement P314. These detailed hazards underscore the impor‑ tance of implementing rigorous safety measures and protocols to minimize the risks associated with handling 4‑Hydroxy‑TEMPO effectively, prioritizing the well‑being of individuals, and ensuring environmental safety [1–3].

FIRST AID MEASURES In the event of exposure to 4‑Hydroxy‑TEMPO (tempol), it’s crucial to respond promptly and effectively to mitigate potential health risks. Inhalation of tempol dust should be immediately addressed by moving the affected indi‑ vidual to an area with fresh air to prevent further exposure. Simultaneously, contacting a physician or seeking medical attention is imperative to assess and address any respiratory symptoms or discomfort, ensuring appropriate treatment and monitoring. For skin contact, prompt action involves removing contaminated clothing and thoroughly rinsing the affected area with water or taking a shower to remove any traces of the compound. This step helps mini‑ mize skin irritation and prevents potential absorption of tempol through the skin. In cases of eye contact, rinsing the eyes with water for several minutes is essential to flush out the compound and alleviate irritation. Contacting an ophthalmologist for further evaluation and treatment is advisable to ensure

168  Chemical and Clinical Applications of Tempol proper care for the eyes. If contact lenses are worn, they should be promptly removed to facilitate thorough rinsing of the eyes. In the unlikely event of ingestion, drinking water can help dilute the compound in the digestive sys‑ tem. However, it’s crucial to seek immediate medical attention and consult a physician for further guidance and evaluation. These detailed response pro‑ cedures emphasize the importance of swift and appropriate action to address exposure to 4‑Hydroxy‑TEMPO, prioritizing the well‑being and safety of the individuals involved [1–3].

HANDLING AND STORAGE To ensure safe handling and storage of 4‑Hydroxy‑TEMPO (tempol), it’s vital to adhere to specific precautions and storage conditions. When handling the compound, precautions should be taken to avoid inhalation of any dust par‑ ticles that may be generated. This can be achieved by working in a well‑ven‑ tilated area or using appropriate respiratory protection if necessary. Adequate ventilation helps disperse any potential airborne particles, reducing the risk of inhalation exposure to tempol. Additionally, during storage, it’s essential to keep the container tightly closed to prevent any potential release of the compound into the surrounding environment. This not only maintains the integrity of the product but also minimizes the risk of accidental exposure. Moreover, ensuring the storage area remains dry is crucial for preserving the stability and quality of tempol. Moisture can lead to degradation of the compound, compromising its effectiveness and safety. The recommended storage temperature, as indicated on the product label, should be strictly fol‑ lowed to maintain the stability of tempol. Deviations from the recommended temperature range may impact the compound’s properties and shelf life. By implementing these precautions for safe handling and adhering to the speci‑ fied storage conditions, the risk of exposure and potential hazards associated with 4‑Hydroxy‑TEMPO can be effectively minimized, ensuring the safety of personnel and maintaining the quality of the compound [1–3].

TOXICOLOGICAL INFORMATION 4‑Hydroxy‑TEMPO (Tempol) exhibits specific acute toxicity and environ‑ mental impact characteristics that necessitate appropriate safety measures

13  •  Safe handling and disposal of 4-HT   169 and environmental considerations. In terms of acute toxicity, the compound demonstrates moderate toxicity when ingested orally, with an LD50 (Lethal Dose, 50%) in rats recorded at 1,053 mg/kg. However, its dermal LD50 in rats is greater than 2,000  mg/kg, indicating lower toxicity through skin contact. Despite causing slight skin irritation, tempol can lead to serious eye damage upon contact, underscoring the importance of eye protection during handling. Notably, tempol does not exhibit sensitization potential according to the Buehler Test in guinea pigs. Regarding environmental impact, tempol poses risks to aquatic organisms. It displays toxicity to fish (Danio rerio) with an LC50 (Lethal Concentration, 50%) of 545 mg/L over 96 hours. Similarly, it exhibits toxicity to daphnia (Daphnia magna) with an EC50 (Effective Concentration, 50%) of 54 mg/L over 48 hours. Additionally, tempol impacts algae (Desmodesmus subspicatus) with an ErC50 (Effective Concentration, 50%) of 1,038 mg/L over 72 hours. These findings underscore the importance of preventing tempol from entering aquatic environments to avoid adverse effects on aquatic life. Furthermore, tempol is classified as not readily biodegradable, indicating its persistence in the environment. Therefore, measures should be taken to minimize its release and mitigate environmental impact. In the event of a fire involving tempol, firefighting measures should include using water, foam, CO2, or dry powder to extinguish the fire. However, inhalation of combustion prod‑ ucts should be avoided due to potential respiratory hazards [1–3].

FIRST AID MEASURES In the event of inhalation of TEMPOL, the affected individual should be moved to an area with fresh air immediately. Fresh air helps to alleviate respiratory discomfort and prevent further inhalation exposure. For skin contact, the contaminated clothing should be promptly removed to prevent prolonged exposure, and the affected skin should be thoroughly rinsed with water. This step helps to remove any traces of the compound from the skin’s surface and minimize the risk of skin irritation or absorption. In cases of eye contact, thorough rinsing with water is essential to flush out the compound and alleviate irritation. Seeking medical attention promptly is advised to ensure proper evaluation and treatment of any eye injuries or discomfort. If TEMPOL is swallowed, drinking water can help dilute the compound in the digestive system. However, it’s crucial to consult a physician immediately for further guidance and evaluation to address any potential health effects [1–3].

170  Chemical and Clinical Applications of Tempol

FIREFIGHTING MEASURES In the event of a fire involving TEMPOL, suitable firefighting methods include using water, foam, carbon dioxide, or dry powder to extinguish the fire. These extinguishing agents help to suppress the flames and cool the burning mate‑ rial effectively. However, it’s important to note that hazardous combustion gases may evolve during the fire, posing respiratory hazards to firefighters and nearby individuals. Therefore, the use of self‑contained breathing appa‑ ratus (SCBA) is essential for firefighters to protect against inhalation of harm‑ ful gases and ensure their safety while extinguishing the fire [1–3].

ACCIDENTAL RELEASE MEASURES In the event of an accidental spill of TEMPOL, immediate actions should be taken to minimize exposure and prevent environmental contamination. Avoiding inhalation and direct contact with the substance is paramount to reduce the risk of adverse health effects. Adequate ventilation should be ensured to disperse any vapors and minimize exposure to airborne particles. Additionally, covering drains and containing the spill using appropriate absorbent materials help prevent the spread of the compound and minimize its impact on the envi‑ ronment. Careful cleaning of the affected area should be conducted to remove any spilled material thoroughly. Proper disposal of the cleanup materials and any contaminated items is essential to prevent further environmental contami‑ nation. By following these measures, the risk of exposure to TEMPOL and its potential environmental impact can be effectively mitigated [1–3].

CONCLUSION In conclusion, the safe handling, storage, and disposal of 4‑Hydroxy‑TEMPO (tempol) are critical considerations in pharmaceutical and research settings. This compound, valued for its radical scavenging and nitric oxide spin trap‑ ping properties, offers diverse applications in biochemical and physiological studies. To ensure safety, efficacy, and compliance with pharmaceutical regu‑ lations, meticulous adherence to safety protocols is essential. Proper storage

13  •  Safe handling and disposal of 4-HT   171 conditions, including selecting suitable containers, guarding against light and heat exposure, and maintaining a dry environment at a regulated tempera‑ ture of −20°C, are imperative to preserve the compound’s integrity. Disposal procedures must adhere to local regulations for hazardous waste, involving appropriate containerization, labeling, and coordination with authorized waste management facilities. Understanding the physical properties of tem‑ pol, such as its crystalline form, melting point, solubility, and density, informs safe handling practices. Moreover, awareness of its hazards, including acute toxicity risks, eye damage potential, and environmental impact, underscores the importance of implementing rigorous safety measures and precautionary protocols. In the event of exposure, prompt and appropriate first aid mea‑ sures are crucial to mitigate potential health risks. Firefighting and acciden‑ tal release measures further emphasize the importance of preparedness and swift action to minimize exposure and prevent environmental contamination.

REFERENCES 1. Occupational Safety and Health Administration (OSHA). “Chemical Hygiene Plan.” OSHA Standard 29 CFR 1910.1450. https://www.osha.gov/lawsregs/ regulations/standardnumber/1910/1910.1450 2. American Chemical Society (ACS). Guidelines for Chemical Laboratory Safety in Academic Institutions. 8th ed.; ACS: Washington, DC, 2016. 3. Food and Drug Administration (FDA). “Guidance for Industry: Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients.” FDA, 2016. https://www.fda.gov/media/121512/download